Peptides for Assisting Delivery Across the Blood Brain Barrier

ABSTRACT

The present invention provides compositions and methods useful for delivering agents to target cells or tissues, for example nerve cells and other cells in the central nervous system. The compositions and methods are useful for delivering agents across the blood-brain barrier. The present invention also provides methods of using the compositions provided by the present invention to deliver agents, for example therapeutic agents for the treatment of neurologically related disorders.

CROSS REFERENCED TO RELATED APPLICATIONS

This Application is a Continuation Application of Ser. No. 14/265,939filed Apr. 30, 2014, which is a Continuation Application of Ser. No.12/301,847 filed Nov. 21, 2008, which is a National Phase EntryApplication under 35 U.S.C. § 371 of International ApplicationPCT/US2007/012152, filed May 22, 2007, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 60/802,377filed on May 22, 2006, the contents of each are incorporated herein intheir entirety by reference.

GOVERNMENT SUPPORT

This invention was supported, in part, by National Institutes of Health(NIH) Grant No. U19 AI056900. The government of the United States hascertain rights to the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 30, 2017, isnamed 2017-06-30-Sequence_Listing.txt and is 12,548 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the field of drug deliveryand more specifically to the delivery of agents to the central nervoussystem, and delivery of agents for the treatment of neurologicallyrelated conditions in the central nervous system and other target cells.

BACKGROUND OF THE INVENTION

The blood brain barrier (BBB) is a system-wide membrane barrier thatprevents the brain uptake of circulating drugs, protein therapeutics,RNAi drugs, and gene medicines. Drugs or genes can be delivered to thehuman brain for the treatment of serious brain disease either (a) byinjecting the drug or gene directly into the brain, thus bypassing theBBB, or (b) by injecting the drug or gene into the bloodstream so thatthe drug or gene enters the brain via the transvascular route across theBBB. With intra-cerebral administration of the drug, it is necessary todrill a hole in the head and perform a procedure called craniotomy. Inaddition to being expensive and highly invasive, this craniotomy baseddrug delivery to the brain approach is ineffective, because the drug orgene is only delivered to a tiny volume of the brain at the tip of theinjection needle. The only way the drug or gene can be distributedwidely in the brain is the transvascular route following injection intothe bloodstream. However, this latter approach requires the ability toundergo transport across the BBB. The BBB has proven to be a verydifficult and stubborn barrier to traverse safely.

The traditional approach to delivery of drugs across the BBB is called“BBB disruption”. One of the earliest techniques tried is the transientdisruption of the barrier (BBBD) by infusing hyperosmolar solutions,which sucks water out of capillary endothelial cells, thereby shrinkingthem to open the gaps [47-49]. Another approach to disrupt BBB is theuse of bradykinin receptor agonists, such as the compound RMP7(Cereport, Alkermes), which binds to the receptors on the surface ofendothelial cells and kicks off a biochemical cascade that loosens thetight junctions [50]. However, none of these methods is very effectiveand moreover, they suffer from the drawback that BBB disruption alsoallows the non-specific entry of other potentially brain-toxic moleculessuch as serum albumin. Accordingly, this approach has not gainedwidespread clinical acceptance.

Transvascular approach provides the most ideal noninvasive means totreat neurological diseases. If not for the BBB, the capillaries, whichstretch for over 400 miles in the brain and encase virtually every braincell, would offer the most promising delivery approach [46]. The mostpromising transvascular approach to brain is to use transportermolecules since this allows delivery of specific molecules withoutdisrupting the BBB [46]. A peptidomimetic mAb, such as against thetransferrin receptor can be used as a molecular “Trojan horse” to ferryany attached drug or gene across the BBB. Recently, great progress hasbeen made in brain delivery by combining the antibody targetingtechnology with siRNA encapsulation within liposomes [46]. The problemsassociated with the use of conventional cationic polyplexes, such as theaggregation of the DNA and sequestration in the lung and liver can beeliminated if the DNA is encapsulated in the interior of a nanocontainersuch as a liposome or a polymeric nanoparticle. If the surface of theliposome is conjugated with polyethylene glycol or hyaluron, this makesthe liposome stable in blood with prolonged blood residence times. Ifthe tips of PEG or hyaluron are conjugated with a BBB molecular “Trojanhorse”, such as anti-transferrin receptor antibody, this immunoliposomeis effectively delivered across the BBB. This system has been used todeliver reporter genes with success in the rat, mice and monkey brains[76-81]. Recently, this technology has also been used to deliver shRNAsto target specific genes in brain tumors in mice as well as monkeys [37,82]. Thus, this method seems optimal to introduce shRNA encoding vectorsas well as synthetic siRNA duplexes.

SUMMARY OF THE INVENTION

The inventors have discovered a method to deliver agents across theblood brain barrier (BBB). As disclosed herein, one embodiment of thepresent invention relates to methods to deliver agents across the bloodbrain barrier. In another embodiment, the present invention providesmethods to deliver agents to a cell, for example a cell with acetylcholine receptors (AchR) present on the surface of the cell. Forexample, the present invention provides methods to deliver agents to aCNS cell.

In one aspect, the present invention relates to a composition comprisinga targeting agent, wherein targeting agent is conjugated with a carrierparticle, and an agent, herein termed an “effector agent” is associatedwith the carrier particle.

In some embodiments, the targeting agent is an RVG peptide or aderivative or a variant thereof. In alternative embodiments, targetingagents include, for example insulin, transferrin, insulin like growthfactor (IGF), leptin, low density lipoprotein (LDL) and fragments orpeptidomimetics or derivatives thereof.

In some embodiments, the carrier particle is, for example, a lyposomalor polymeric nanoparticles, for example a liposome, polyarginine,protamine, or a cyclodextrin-based nanoparticle. In alternativeembodiments, the carrier particle is a cell permeable agent, for examplea cell permeable agent, for example but not limited to, 11dR, 9dR orTAT-HIV or a fragment thereof.

In some embodiments, where the carrier particle is, for example, alyposomal or polymeric nanoparticles, for example a liposome,polyarginine, protamine, or a cyclodextrin-based nanoparticle, thecarrier particle can optionally further comprise cell permeable agents,for example but not limited to, polymeric arginine residues of variouslengths such as 11R or 9R as disclosed herein or TAT-HIV or fragmentsthereof. In other embodiments, the carrier particle can optionallyfurther comprise additional targeting agents, for example, inembodiments where the carrier particle is conjugated to an RVG peptide,the carrier particle can also comprise additional targeting agents, forexample insulin, transferrin, insulin like growth factor (IGF), leptin,low density lipoprotein (LDL) and fragments or peptidomimetics orderivatives thereof.

One aspect of the present invention relates to a method for preparing aneffector agent for delivery to a cell, comprising associating aneffector agent with a carrier particle, wherein the carrier particle isassociated with a rabies virus glycoprotein (RVG) peptide comprising SEQID NO:13 or a variant, fragment or derivative thereof, wherein the RVGpeptide binds to an acetylcholine receptor (AChR) present on the surfaceof the cell. In such embodiments, a derivative or variant thereofcomprises a conservative amino acid substitution.

In another aspect, the present invention relates to a method fordelivering an effector agent across the blood-brain barrier of asubject, the method comprising administering to the subject acomposition comprising a rabies virus glycoprotein (RVG) peptidecomprising SEQ ID NO:13 or a variant, fragment or derivative thereof,wherein the RVG peptide is attached to a carrier particle, and whereinthe effector agent is associated with the carrier particle.

A further aspect of the present invention relates to a method fordelivering an effector agent to a cell of the CNS, the method comprisingcontacting the cell with a rabies virus glycoprotein (RVG) peptidecomprising SEQ ID NO:13 or a variant, fragment or derivative thereof,wherein the RVG peptide is attached to a carrier particle, and whereinthe effector agent is associated with the carrier particle, and the RVGpeptide binds to binds to an acetylcholine receptor (AChR) present onthe surface of the cell of the CNS.

A further aspect of the present invention relates to a composition fortargeted delivery of an effector agent to a cell, wherein thecomposition comprises at least one rabies virus glycoprotein (RVG)peptide comprising SEQ ID NO:13 or a variant, fragment or derivativethereof, and at least one carrier particle attached thereto, wherein theeffector agent is associated with the carrier particle.

In some embodiments, carrier particle comprises a cell permeable agent,for example a cell permeable peptide. In some embodiments, the cellpermeable peptide is a polymeric arginine residue of various lengths,such as 11 residues (termed “11dR” or “11R” herein) or 9 residues(termed “9R” herein), or 7 or 5 residues in length, as disclosed herein.

In some embodiments the carrier particle comprises a liposome,polyarginine, protamine, or a cyclodextrin-based nanoparticle.

In some embodiments, the effector agent is delivered to a cell, forexample a cell is located inside the blood brain barrier, for example acentral nervous system cell. Examples of central nervous system cellsinclude, for example but not limited to neuron, neuronal cell, braincells, glial, astrocyte or neuronal supporting cells. In someembodiments, the cell comprises acetyl choline receptor (AchR) or ahomologue or fragment thereof, for example the cell comprises the asubunit of AchR or a fragment or homologue thereof. In some embodiments,the cell comprises the α1 and/or α7 subunit of AchR or a fragment orhomologue thereof.

In some embodiments, the cell is present within a subject, for example amammalian subject, for example a human subject. In alternativeembodiments, the cell is ex vivo, and in further embodiments, the cellis in a biological sample, for example in vitro.

In some embodiments, an effector agent is a nucleic acid or a nucleicacid analogue, for example but not limited to a DNA or RNA, for examplesiRNA, mRNA, tRNA, miRNA, strand template RNA (stRNA), shRNA, oranalogues or combinations thereof. In some embodiments, an effectoragent is a nucleic acid analogue, for example but not limited toantisense nucleic acids, oligonucleic acids or oligonucleotides, peptidenucleic acid (PNA), pseudo-complementary PNA (pcPNA), locked nucleicacid (LNA) or derivatives or analogues thereof. In some embodiments, theeffector is a miRNA mimetic and in alternative embodiments, the effectoris an antigomir, an oligonucleotide for silencing and/or inhibitingendogenous miRNA.

In alternative embodiments, the effector agent is a small molecule, andin alternative embodiments the effector agent is a protein or peptide,or the effector agent is a peptidomimetic, aptamer, antibody or proteinor variant thereof. In some embodiments, the effector agent is anavimer, which is a peptide having multi-target site recognitioncapability.

In further embodiments, the effector agent is a therapeutic agent and/ordiagnostic agent and/or an imaging agent.

In some embodiments, the carrier particle further comprises anadditional targeting agent, wherein the targeting agent is a blood brainbarrier targeting agent. Examples of such blood-barrier targeting agentsinclude, for example but are not limited to, insulin, transferrin,insulin like growth factor (IGF), leptin, low density lipoprotein (LDL)and fragments or peptidomimetics or derivatives thereof. In someembodiments, additional targeting agents can be avimers for targetingreceptors on the surface cells of the BBB, for example for targetingreceptors for insulin, transferrin, insulin like growth factor (IGF),leptin, low density lipoprotein (LDL).

In some embodiments, the composition comprises an effector agentassociated to a carrier particle, where the carrier particle isconjugated to a targeting agent (for example an RVG peptide). Thecomposition is useful to deliver effector agents across the blood brainbarrier in a subject. In some embodiments, the composition can furthercomprise a pharmaceutically acceptable carrier.

In further embodiments, the composition as disclosed herein is useful asa medicant for central nervous system disorders.

In some embodiments, administration of such a composition can be by anysuitable route, for example but not limited to subcutaneous,intravenous, intracranial, or oral administration. In furtherembodiments, administration is, for example but not limited to,parenteral, intranasal, intracranial or intravenous.

Another aspect of the present invention relates to a method fordelivering an effector agent to a cell expressing the alpha subunit ofthe acetylcholine receptor, the method comprising contacting the cellwith an RVG peptide comprising SEQ ID NO:13 or a variant, fragment orderivative thereof, wherein the RVG peptide is attached to a carrierparticle, wherein the effector agent is associated with the carrierparticle. In some embodiments, the cells are in vitro, and in someembodiments the cells are in vivo or ex vivo.

Another aspect of the present invention relates to a method fordelivering an agent across the blood-brain barrier of a subject, themethod comprising administering to the subject a composition comprisinga targeting agent, wherein the targeting agent is attached to a carrierparticle, and wherein the agent is associated with the carrier particle.In some embodiments, the targeting agent is an RVG peptide of SEQ IDNO:13 or a variant, fragment or derivative thereof, and in alternativeembodiments, the targeting agent is, for example but not limited to,insulin, transferrin, insulin like growth factor (IGF), leptin, lowdensity lipoprotein (LDL) and fragments or peptidomimetics orderivatives thereof.

The present invention provides a method for delivery of an agent to thecentral nervous system (CNS) of a host. The method comprisesadministering to the host an agent, wherein the agent comprises an RVGpeptide comprising SEQ ID NO:13 13 or a variant, fragment or derivativethereof, wherein the RVG peptide is attached to a carrier particle and atherapeutic agent is associated with the carrier particle.

The invention further provides a targeted delivery compositioncomprising an RVG peptide, wherein the RVG peptide is attached to acarrier particle. The invention still further provides the targeteddelivery composition wherein an effector agent is a therapeutic agent,which is associated with the carrier particle.

In one embodiment, the carrier particle is a lyposomal or polymericnanoparticle, for example a liposome, a polyarginine peptide, aprotamine or a cyclodextrin-based nanoparticle.

In one embodiment, the effector agent or therapeutic agent is a nucleicacid, e.g., siRNA, shRNA, stRNA, miRNA or DNA or nucleic acid analogues,antigomers or derivatives thereof. In another embodiment, thetherapeutic agent is a small molecule. In yet another embodiment, thetherapeutic agent is a protein, e.g., an enzyme, an antibody,peptidiomimetic, aptamer, avimer and derivatives thereof.

In one embodiment, the carrier particle further comprises a blood brainbarrier targeting agent in addition to the RVG peptide.

In one embodiment, the agent is further administered with apharmaceutically acceptable carrier. In one embodiment, theadministration is parenteral, intranasal, intracranial or intraocular.

The invention further provides a method for targeted delivery of anagent to a cell expressing the alpha subunit of the acetylcholinereceptor. The method comprises administering the agent to the cell. Theagent comprises an RVG peptide comprising SEQ ID NO: 13 or a fragment,derivative or variant thereof, wherein the RVG peptide is attached to acarrier particle, wherein a therapeutic agent is associated with thecarrier particle. In one embodiment, the cell is in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pLL3.7 lentiviral vector.

FIG. 2 shows efficient transduction and intracellular processing ofshRNA. BHK 21 cells were transduced with lentiviruses and analyzed byflow cytometry for GFP expression (left) and tested for endogenousFvE^(J) specific siRNA production by Northern blotting (right).

FIG. 3 shows FvE^(J) shRNA inhibits JEV replication. Transduced BHK 21cells were infected with Japanese Encephalitis virus (JEV) and viralreplication analyzed by flow cytometry 2 days later using a JEV-specificantibody.

FIG. 4 shows degradation of viral RNA by FvE^(J) shRNA. RNA fromtransduced and infected cells was probed with a JEV cDNA probe.

FIG. 5 shows FvE^(J) shRNA protects against JEV. Mock, control Luc orFvE^(J) shRNA injected mice were challenged with JEV and observed formortality.

FIG. 6 shows absence of brain pathology in FvE^(J) shRNA-treated mice.Mice injected with Luc shRNA (left) or FvE^(J) shRNA (right) wereinfected with JEV for 5 days and the brain sections examined forhistopathology. Shown are low magnification (×20) (top panels) and highmagnification (×400) (bottom panels).

FIGS. 7A-7B show the absence of virus in FvE^(J) shRNA-treated mice.Mice were treated as in FIG. 5. Panel 7A shows FACs analysis of braincells administered with Luc-siRNA or FvE-siRNA. Panel 7B shows brainhomogenates from animals administered with Luc-siRNA, or FvE-siRNA andtitered for viral titre of JEV.

FIGS. 8A-8B show siFvE^(J) siRNA can protect against JE in vitro as wellas in vivo. FIG. 8A shows neuro 2A cells transfected with siLuc (greyfilled) or with siFvE^(J) siRNA in lipofectamine (broken line) orcomplexed with JetSI/dope (solid line) were infected with JEV for 2 daysbefore staining. FIG. 8B shows mice (5/group) were infected with JEV andtreated with control or siFvE^(J) siRNA after 30 min or 6 h afterinfection.

FIGS. 9A-9B show siFvE^(JW) siRNA protects against both JapaneseEncephalitis virus (JEV) and West Nile Virus (WNV) in vitro and in vivo.FIG. 9A shows neuro 2A cells treated with siLuc or siFvE^(JW) siRNAcomplexed with JetSI/dope were infected with JEV or WNV for 2 daysbefore staining. FIG. 9B shows mice (10/group) were infected with JEV(left) or WNV (right) and treated with control siLuc or siFvE^(JW) siRNAafter 30 min or 6 h after infection and followed for survival over time.

FIG. 10 shows RVG pseudotyping allows neuronal cell specific targeting.HeLa or BHK-21 or Neuro 2a cells were infected with PLL3.7 lentivirusespseudotyped with either VSV-G or RVG and examined for GFP fluorescence 2days later.

FIG. 11 shows RVG pseudotyping enhances shRNA effectiveness. Mice wereinjected with indicated doses of lentiviruses psuedotyped with eitherVSV-G or RVG, challenged with JEV and observed for mortality.

FIG. 12 shoes RVG peptide specifically binds neuronal cells. Neuro 2aand HeLa cells were sequentially incubated with RVG-Bio or ScrambledRVG-Bio and SAPE and examined by flow cytometry.

FIG. 13 shows α-bungarotoxin inhibits RVG peptide binding. RVG peptidebinding. Neuro 2a cells were stained with RVG-Bio peptide in thepresence or absence of a-bungarotoxin then tested for RVG peptide (10-7M) binding.

FIG. 14 shows RVG peptide binds mouse brain cells. Freshly isolatedbrain and spleen cells were stained with RVG-Bio and SAPE.

FIG. 15 shows intravenously injected RVG peptide binds brain cells. Micewere given a control peptide or RVG-Bio intravenously and 2 h later,brain cells stained with SAPE.

FIG. 16 shows RVG-TAT peptide can deliver DNA vector to neuronal cells.Neuro 2a or BHK-21 cells were transduced with pLL3.7 DNA bound toRVG-TAT and examined for GFP expression 2 days later.

FIG. 17 shows RVG-11dR peptide delivers both DNA and siRNA. Neuro 2acells were transduced with pLL3.7 DNA vector (left) or FITC labeledsiRNA (right) bound to RVG-11dR and examined for GFP or FITCfluorescence 2 days later.

FIG. 18 shows RVG-11dR delivered siRNA is functional. Neuro 2a cellswere first transfected with GFP DNA and then treated with GFP siRNAalone or bound to RVG-11dR and examined 2 days later.

FIG. 19 shows immunoliposomes for targeted delivery of siRNA. ActivatedCD4 T cells were incubated with CD4 siRNA-encapsulated hyaluranliposomes coated with LFA-1 (AL-57) or an isotype control (IgG1)antibody and examined for CD4 expression 2 days later.

FIGS. 20A-20B show RVG binds specifically to the neuronal cell line,Neuro 2a and the binding is inhibited by α-bungarotoxin. FIG. 20A showsHeLa and Neuro 2a cells incubated with biotinylated RVG peptide andexamined for peptide binding by staining with streptavidin-PE (SAPE).Control peptide did not bind to either cell type while RVG binding wasdetected exclusively on Neuro 2a cells. FIG. 20B shows RVG binding toNeuro 2a cells was measured in the presence of indicated concentrationsof α-bungarotoxin. RVG peptide was used at 2 μM.

FIG. 21 shows RVG can be detected in the mouse brain after intravenousinjection. Mice were injected i.v. with 100 μg of biotinylated RVG orthe control peptide and 2 h later, single cell suspensions of brainexamined by flow cytometry after internal staining with SAPE.

FIGS. 22A-22B show CORVUS (a chimeric peptide comprising an RVG peptidefused to a cell penetrating peptide) binds and delivers siRNA intoneuronal cells. FIG. 22A shows 100 pmole of siRNA was complexed withCORVUS or control peptide at the indicated molar ratios and binding ofsiRNA was assessed by gel mobility retardation on 2% agarose gels. FIG.23B shows peptides incubated with 100 pmoles FITC-labeled siRNA at theindicated concentrations for 10 min and then added to Neuro 2a cells inculture. siRNA uptake was assessed 16 h later. Control peptide was usedat 25 μM. A 10:1 molar ratio of peptide to siRNA was deemed optimal.

FIGS. 23A-23B show CORVUS delivered siRNA is functional. Peptides weremixed with 200 pmole of anti-GFP siRNA at a 10:1 molar ratio and addedto Neuro 2a cells stably expressing GFP. FIG. 23A shows GFP expressionlevels were monitored 60 h later. FIG. 23B shows CORVUS was found to beas efficient as Lipofectamine 2000™ in silencing GFP expression.

FIGS. 24A-24E show CORVUS (RVG-9R) can deliver siRNA to brain cellsafter i.v. injection in mice. Mice were injected twice, 6 h apart,intravenously in the tail vein with 50 μg of FITC-labeled siRNAcomplexed to peptides at a 10:1 molar ratio. FIG. 24A-E shows organswere harvested 16 h after the last injection and single cell suspensionsanalyzed for the presence of siRNA FITC. FIG. 24A shows control brainsamples, Fog 24B shows liver with CORVUS or control peptide, FIG. 24Cshows brain from CORVUS and control treated samples, and FIG. 24D showsspleen with CORVUS or control peptide. FIG. 24E shows siRNA was detectedspecifically in the brain tissue of mice treated with CORVUS. Controlpeptide did not induce any uptake of siRNA.

FIGS. 25A-25B show transvascular delivery of GFP siRNA complexed toCORVUS specifically knocks down GFP expression in GFP-Tg mice. Micetransgenic for GFP were intravenously administered 50 μg anti-GFP siRNAcomplexed to either CORVUS or control peptides 3 times at 16 hintervals. FIG. 25A shows organs harvested 60 h after the last treatmentand single cell suspensions analyzed for GFP expression levels. FIG. 25Bshows filled histograms represent FACS plots with wild-type mice.

FIGS. 26A-26E show a short RVG peptide binds to neuronal cells in vitroand in vivo. FIG. 26A shows Neuro 2a and HeLa cells (inset) wereincubated with biotinylated RVG or RV-MAT peptides, stained with SAPEand examined by flow cytometry. FIG. 26B shows Peptide binding was alsotested using indicated cell lines. RVMAT did not bind any of the celllines (not shown). FIG. 26C shows Neuro 2a cells were stained withbiotinylated RVG in the absence (red histogram) or presence ofdecreasing concentrations of BTX (grey histograms). FIG. 26D showsfreshly isolated mouse brain and spleen cells were tested for peptidebinding. FIG. 26E shows mice were injected iv with biotinylated RVG orRV-MAT peptide and 4 h later, isolated brain cells stained with SAPE.

FIGS. 27A-27D show an RVG-9R peptide binds and delivers siRNA toneuronal cells in vitro resulting in gene silencing. FIG. 27A showsmobility of free or peptide-complexed siRNA was analyzed by agarose gelelectrophoresis. FIG. 27B shows Neuro 2a cells were examined for uptakeof FITC-siRNA complexed with RVG-9R at the indicated concentrations.FIG. 27C shows Neuro 2a and HeLa (inset) cells were examined for uptakeof FITC-siRNA complexed with RVG-9R or RV-MAT-9R peptides at 1:10 molarratio. Lipofectamine transfection was used as positive control. FIG. 27Dshows Neuro 2a cells stably expressing GFP were transduced with GFPsiRNA complexed to RVG-9R or RV-MAT-9R peptides and GFP silencing tested2 days later. A representative histogram and cumulative data from 3independent experiments (inset) are shown.

FIGS. 28A-28B show RVG-9R enables transvascular delivery of siRNA to theCNS. FIG. 28A shows mice were injected iv with FITC-siRNA/peptidecomplexes and uptake by brain, spleen and liver cells examined by flowcytometry. Representative histograms (top) and cumulative data (bottom)are shown. FIG. 28B shows coronal sections of brain fromFITC-siRNA/RVG-9R injected mice (n=6) were stained with anti-FITCantibody and examined by fluorescent microscopy. Images of FITC-positivecells in the cortex, striatum and thalamus at lower (left panel) andhigher magnification of boxed regions (middle panel) are shown. Rightpanel shows images from control Ig stained brain sections at the highermagnification. Scale bar=200 μm.

FIGS. 29A-29D show brain-specific gene silencing by iv injection ofRVG-9R/siRNA complex. FIG. 29A shows GFP Tg mice were iv injected withGFP siRNA/peptide complexes and their brain, spleen and liver cellsanalyzed for GFP expression. Representative histograms (top) andcumulative data (bottom) are shown. Dotted lines in the histogramsrepresent cells from wild type mice. FIG. 29B shows Balb/c mice ivinjected with SOD1 siRNA/peptide complexes and their brain, spleen andlivers examined for SOD1 mRNA (top) and protein levels (bottom). Thenumbers below the western blot represent the ratios of band intensitiesof SOD-1 normalized to that of β-actin. FIG. 29C shows small RNAsisolated from different organs of RVG-9R/SOD1 siRNA injected mice wereprobed with siRNA sense strand oligo. Antisense strand oligo was used aspositive control (first and last lanes). FIG. 29D shows mice iv injectedwith SOD siRNA bound to RVG-9R and the duration of gene silencingdetermined by quantitation of SOD1 mRNA levels (top) and SOD1 proteinenzyme activity (bottom) on indicated days after siRNA administration.

FIGS. 30A-30C show iv treatment with antiviral siRNA/RVG-9R complexprotects mice against JEV encephalitis. FIG. 30A shows JEV-infected micewere treated iv with siLuc or siFvEJ complexed to either RVG-9R orRVMAT-9R daily for 4 days and monitored for survival. FIG. 30B shows RNAisolated from the brains of RVG-9R/siFvEJ treated mice were examined forthe presence of siRNA antisense strand by Northern blotting. Antisensestrand of siFvEJ served as positive control. FIG. 30C shows Balb/c micewere injected iv with siFvEJ bound to RVG-9R peptide and 7 h later,their serum samples tested for IFN levels by ELISA. Theimmunostimulatory βgal 728 siRNA complexed with RVG-9R or lipofectaminewas used as positive control.

FIG. 31 shows RVG-9R binding confers partial protection from serumnucleases. Naked and RVG-9R-complexed siRNA were incubated with sera at37° C. and aliquots taken at indicated times digested with proteinase K,electrophoresed on 15% polyacrylamide gels and visualized with SYBR goldstaining. The position of intact siRNA is indicated.

FIGS. 32A-32B show an RVG-9R siRNA complex does not induce antibodies orinflammatory cytokines. FIG. 32A shows mice injected with siRNAcomplexed to RVG-9R or for positive control, with the immunogenicTNP-KLH-biotin peptide on days 0, 3, 10 and 22 and serum samplescollected on days 21 and 30 tested for the presence of antibodies to RVGor biotin by ELISA. FIG. 32B shows sera obtained 1 day after the 4_(th)RVG-9R/siRNA injection were tested for the indicated panel of secretedcytokines and chemokines in an ELISA assay. Sera from LPS injected miceserved as positive control. Asterisks indicate statistically significantdifferences.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

A major bottleneck in harnessing the potential of RNAi for clinical useis the lack of suitable delivery methods. Delivery is particularlydifficult in the CNS and the methods used for systemic delivery such asintravenous hydrodynamic injection [42, 43] and intravenous (IV or iv)injection of siRNA or shRNA vectors complexed with lipofectamine orpolyethyleneimine [44] are unlikely to work for delivery to the CNSbecause of the presence of BBB. Thus, the only available method for CNSdelivery at present is local sterotaxic injection of nonreplicatingviral vectors and siRNA [61]. One problem with these approaches is theextremely limited spread, confining delivery to a small area at the siteof injection. Thus, delivery methods to ensure a more extensive spreadof the delivered si/shRNAs for its efficacy in situations like tumorsand intracranial infections are needed. The inventors have discovered apeptide derived from Rabies virus glycoprotein (RVG) can specificallytarget neuronal cells. This peptide has previously been shown tocompetitively inhibit α-bungarotoxin binding to the nicotinicacetylcholine receptor α7 subunit [94, 95, 102]. Acetylcholine receptorα7 subunit is widely expressed by many cell types in the brain includingthe neurons, astrocytes and glia cells and it is also expressed by thebrain capillary endothelial cells [98].

Accordingly, in one embodiment the present invention provides methods todeliver agents to the brain or spinal cord, wherein at least one agentis associated to RVG peptide as disclosed herein. In some embodiments,the RVG peptide is associated with a carrier particle, for example alyposomal or polymeric nanoparticles, such as a liposome, and the agentis associated with the carrier particle. In some embodiments, thecarrier particle is a cell permeable agent. In further embodiments, thecell permeable agent is a cell permeable peptide, for example polymericarginine residues of various lengths such as 11dR or 9R as disclosedherein, or TAT. In further embodiments the RVG peptide is conjugated toother targeting agents, or the carrier particle is conjugated toadditional targeting genes.

The inventors have discovered that the RVG peptide results in extensivespread of agents in the central nervous system, thus results in deliveryof an agent to sites distal to the site of administration, for exampledistal to the site of intracranial or intraparemchal injection.Moreover, in one embodiment, the RVG peptide facilitates the crossing ofan agent across the BBB. In some embodiments, the RVG peptide isconjugated, for example fused to a cell penetrating peptide (such as forexample but not limited to, HIV-TAT, or polymeric arginine residues ofvarious lengths such as 9R or 11dR as disclosed herein) or combined witha brain endothelial cell transporter (such as transferrin or transferrinreceptor antibody), and thus, facilitates brain delivery by anoninvasive intravenous approach. In some embodiments, the effectoragents are therapeutic agents.

Therapeutic agents for which are, for example nucleic acids such assiRNA, miRNA and shRNAs effector agents can be expressed via vectorsoffer the advantage of long term expression which can be desirable insome situations like in the treatment of neurological disorders,neurodegenerative diseases and cancer. Synthetic siRNAs offer adrug-like approach for transient gene silencing. Moreover, in thenon-dividing cells of the CNS, the effect is prolonged, e.g., 3 weeks.Alternatively, the RVG delivery method can be used in conjunction withtherapeutic agents that are not RNAi agents, such as but not limited tosmall molecules, peptides, antibodies, avimers, nucleic acid analogues,antigomers, miRNA mimetics or any other agent that is compatible withdelivery by means of the carrier particles associated with an RVGpeptide as disclosed herein.

As disclosed herein, the present invention is based, in part, on thediscovery that peptides derived from the rabies virus glycoprotein areuseful as targeting moieties to deliver agents to cells expressing the asubunit of the acetylcholine receptor or a homologue thereof and forexample neuronal cells. In some embodiments, the neuronal cells are in asubject (i.e. in vivo), and in some embodiments the neuronal cells areex vivo or are cultured neuronal cells, for example in vitro such asprimary neuronal cultured cells. In some embodiments, the neuronal cellsare neuronal precursor or neuronal progenitor cells, such as neuronalprogenitor stem cells that express the a subunit of the acetylcholinereceptor or a homologue thereof.

Accordingly, the present invention is also directed to a method and acomposition for delivering therapeutic compositions to target cells. Inparticular, the invention is directed to a method and a composition fortargeted delivery to target cells protected by the blood brain barrier(BBB). The method utilizes a composition comprising a peptide derivedfrom the Rabies virus glycoprotein (RVG) that is capable of specificallybinding to target cells, but not other cell types. The compositionfurther comprises a carrier particle attached to an RVG peptide asdisclosed herein. The carrier particle can be further associated with aneffector agent, or a therapeutic composition. Thus, the invention isdirected to targeted delivery to target cells by means of an RVGpeptide.

Targeting Agents and Rabies Virus Glycoprotein (RVG) Peptide

The glycoprotein from the neurotropic Rabies virus shows a significanthomology with the snake venom alpha neurotoxin that binds to thenicotinic acetylcholine receptor [91]. In fact, further studies showedthat the acetylcholine receptor is also a Rabies virus receptor [92,93]. Interestingly, an RVG peptide was also found to competitivelyinhibit α-bungarotoxin binding to the acetylcholine receptor [94, 95].However, there has been no indication that the 29 mer RVG peptide (RVG)as disclosed herein facilitates targeted delivery to such cellsexpressing the acetylcholine receptor or that such a peptide canfacilitate passage through the blood brain barrier.

Accordingly, in one embodiment the present invention provides atargeting agent to selectively targets cells expressing the a subunit ofthe acetylcholine receptor, thereby facilitating specific delivery tosuch target cells. In one embodiment of the present invention, atargeting agent comprises amino acid residues 173-202 of the RVG:YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 13) or a variant or aderivative or fragment thereof. In further embodiments, the targetingagent is a fragment of SEQ ID NO:13. Such a fragment of SEQ ID NO:13 canbe, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deletedfrom the N-terminal and/or C-terminal of SEQ ID NO:13. Persons ofordinary skill in the art can easily identify the minimal peptidefragment of SEQ ID NO:13 by sequentially deleting N- and/or C-terminalamino acids from SEQ ID NO:13 and assessing the function of theresulting peptide fragment, such as function of the peptide fragment tobind acetylcholine receptor and/or ability to transmit through the bloodbrain barrier as disclosed herein. In some embodiments, a fragment ofSEQ ID NO:13 is any 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16or 15 peptides of SEQ ID NO:13. In some embodiments, a fragment of SEQID NO:13 is less than 15 peptides in length.

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example but not limited to by techniques suchas ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol) or addition of other molecules.

As used herein, “variant” with reference to a polynucleotide orpolypeptide, refers to a polynucleotide or polypeptide that can vary inprimary, secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide). A “variant” of a RGV peptide,for example SEQ ID NO:13 is meant to refer to a molecule substantiallysimilar in structure and function, i.e. where the function is theability to pass or transit through the BBB, to either the entiremolecule, or to a fragment thereof. A molecule is said to be“substantially similar” to another molecule if both molecules havesubstantially similar structures or if both molecules possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif the structure of one of the molecules not found in the other, or ifthe sequence of amino acid residues is not identical.

For example, a variant of an RVG peptide can contain a mutation ormodification that differs from a reference amino acid in SEQ ID NO:13.In some embodiments, a variant of SEQ ID NO:13 is a fragment of SEQ IDNO:13 as disclosed herein. In some embodiments, a variant can be adifferent isoform of SEQ ID NO:13 or can comprise different isomer aminoacids. Variants can be naturally-occurring, synthetic, recombinant, orchemically modified polynucleotides or polypeptides isolated orgenerated using methods well known in the art. Variants can includeconservative or non-conservative amino acid changes, as described below.Polynucleotide changes can result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. Variants can also include insertions,deletions or substitutions of amino acids, including insertions andsubstitutions of amino acids and other molecules) that do not normallyoccur in the peptide sequence that is the basis of the variant, forexample but not limited to insertion of ornithine which do not normallyoccur in human proteins. The term “conservative substitution,” whendescribing a polypeptide, refers to a change in the amino acidcomposition of the polypeptide that does not substantially alter thepolypeptide's activity. For example, a conservative substitution refersto substituting an amino acid residue for a different amino acid residuethat has similar chemical properties. Conservative amino acidsubstitutions include replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, or a threonine with a serine.“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the BBB).Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, the following six groupseach contain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See alsoCreighton, Proteins, W. H. Freeman and Company (1984).) In someembodiments, individual substitutions, deletions or additions thatalter, add or delete a single amino acid or a small percentage of aminoacids can also be considered “conservative substitutions” is the changedoes not reduce the activity of the peptide (i.e. the ability of an RVGpeptide variant to penetrate the BBB). Insertions or deletions aretypically in the range of about 1 to 5 amino acids. The choice ofconservative amino acids may be selected based on the location of theamino acid to be substituted in the peptide, for example if the aminoacid is on the exterior of the peptide and expose to solvents, or on theinterior and not exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986);205-218 and S. French and B. Robson, J. Mol. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants. A variant of a RGV peptide, for example a variantof SEQ ID NO:13 is meant to refer to any molecule substantially similarin structure and function to either the entire molecule of SEQ ID NO:13,or to a fragment thereof. A molecule is said to be “substantiallysimilar” to another molecule if both molecules have substantiallysimilar structures or if both molecules possess a similar biologicalactivity, for example if both molecules are able to penetrate the BBB.Thus, provided that two molecules possess a similar activity, (i.e. avariant of an RVG peptide which can penetrate the BBB similar to that ofthe RVG peptide corresponding to SEQ ID NO:13) are considered variantsand are encompassed for use as disclosed herein, even if the structureof one of the molecules not found in the other, or if the sequence ofamino acid residues is not identical.

As used herein, the term “nonconservative” refers to substituting anamino acid residue for a different amino acid residue that has differentchemical properties. The nonconservative substitutions include, but arenot limited to aspartic acid (D) being replaced with glycine (G);asparagine (N) being replaced with lysine (K); or alanine (A) beingreplaced with arginine (R).

The term “insertions” or “deletions” are typically in the range of about1 to 5 amino acids. The variation allowed can be experimentallydetermined by producing the peptide synthetically while systematicallymaking insertions, deletions, or substitutions of nucleotides in thesequence using recombinant DNA techniques.

The term “functional derivative” and “mimetic” are used interchangeably,and refers to a compound which possess a biological activity (eitherfunctional or structural) that is substantially similar to a biologicalactivity of the entity or molecule its is a functional derivative of.The term functional derivative is intended to include the fragments,variants, analogues or chemical derivatives of a molecule.

The term “fragment” of a peptide or molecule as used herein refers toany contiguous polypeptide subset of the molecule. Fragments of an RGVpeptide, for example fragments of SEQ ID NO:13 useful in the methods asdisclosed herein have the same activity as that of SEQ ID NO:13. Statedanother way, a fragment of an RVG peptide is a fragment of SEQ ID NO:13which can penetrate the BBB and/or bind α acetylcholine receptor as theRVG peptide corresponding to SEQ ID NO:13. Fragments as used hereintypically are soluble (i.e. not membrane bound). Examples of fragmentsof SEQ ID NO:13 include but are not limited to any 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16 or 15 peptides of SEQ ID NO:13. In someembodiments, a fragment of SEQ ID NO:13 is less than 15 peptides inlength.

As used herein, “homologous”, when used to describe a polynucleotide,indicates that two polynucleotides, or designated sequences thereof,when optimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least 70% of the nucleotides,usually from about 75% to 99%, and more preferably at least about 98 to99% of the nucleotides. The term “homolog” or “homologous” as usedherein also refers to homology with respect to structure and/orfunction. With respect to sequence homology, sequences are homologs ifthey are at least 50%, at least 60 at least 70%, at least 80%, at least90%, at least 95% identical, at least 97% identical, or at least 99%identical. The term “substantially homologous” refers to sequences thatare at least 90%, at least 95% identical, at least 97% identical or atleast 99% identical. Homologous sequences can be the same functionalgene in different species.

As used herein, the term “substantial similarity” in the context ofpolypeptide sequences, indicates that the polypeptide comprises asequence with at least 60% sequence identity to a reference sequence, or70%, or 80%, or 85% sequence identity to the reference sequence, or mostpreferably 90% identity over a comparison window of about 10-20 aminoacid residues. In the context of amino acid sequences, “substantialsimilarity” further includes conservative substitutions of amino acids.Thus, a polypeptide is substantially similar to a second polypeptide,for example, where the two peptides differ by one or more conservativesubstitutions.

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

An “analog” of a molecule such as RGV peptide, for example SEQ ID NO:13refers to a molecule similar in function to either the entire moleculeor to a fragment thereof. The term “analog” is also intended to includeallelic, species and induced variants. Analogs typically differ fromnaturally occurring peptides at one or a few positions, often by virtueof conservative substitutions. Analogs typically exhibit at least 80 or90% sequence identity with natural peptides. Some analogs also includeunnatural amino acids or modifications of N or C terminal amino acids.Examples of unnatural amino acids are, for example but not limited to;acedisubstituted amino acids, N-alkyl amino acids, lactic acid,4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine. Fragments andanalogs can be screened for prophylactic or therapeutic efficacy intransgenic animal models as described below.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties can improve themolecule's solubility, absorption, biological half life, etc. Themoieties can alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl.,Easton, Pa. (1990).

The term “substitution” when referring to a peptide, refers to a changein an amino acid for a different entity, for example another amino acidor amino-acid moiety. Substitutions can be conservative ornon-conservative substitutions.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G. U. or 1) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “substantial identity” as used herein denotes a characteristicof a polynucleotide or amino acid sequence, wherein the polynucleotideor amino acid comprises a sequence that has at least 85 percent sequenceidentity, preferably at least 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 18 nucleotide (6 aminoacid) positions, frequently over a window of at least 24-48 nucleotide(8-16 amino acid) positions, wherein the; percentage of sequenceidentity is calculated by comparing the reference sequence to thesequence which can include deletions or additions which total 20 percentor less of the reference sequence over the comparison window. Thereference sequence can be a subset of a larger sequence. The term“similarity”, when used to describe a polypeptide, is determined bycomparing the amino acid sequence and the conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.The term “homologous”, when used to describe a polynucleotide, indicatesthat two polynucleotides, or designated sequences thereof, whenoptimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least 70% of the nucleotides,usually from about 75% to 99%, and more preferably at least about 98 to99% of the nucleotides.

Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan. The terms“homology” or “identity” or “similarity” are used interchangeably hereinand refers to sequence similarity between two peptides or between twonucleic acid molecules. Homology and identity can each be determined bycomparing a position in each sequence which can be aligned for purposesof comparison. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar amino acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, though preferably less than 25% identity with asequence of the present application.

In one embodiment, the term “RVG peptide homolog” refers to an aminoacid sequence that has 40% homology to the full length amino acidsequence of the RVG peptide as disclosed herein, for example the RVGpeptide corresponding to SEQ ID NO:13 as disclosed herein, morepreferably at least about 50%, still more preferably, at least about 60%homology, still more preferably, at least about 70% homology, even morepreferably, at least about 75% homology, yet more preferably, at leastabout 80% homology, even more preferably at least about 85% homology,still more preferably, at least about 90% homology, and more preferably,at least about 95% homology. As discussed above, the homology is atleast about 50% to 100% and all intervals in between (i.e., 55%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482 (1981), which is incorporated by reference herein), bythe homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-53 (1970), which is incorporated by reference herein), by thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. USA 85:2444-48 (1988), which is incorporated by reference herein),by computerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. (See generally Ausubel et al. (eds.), Current Protocols inMolecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which isincorporated by reference herein). The method used is similar to themethod described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53(1989), which is incorporated by reference herein). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), whichis incorporated by reference herein). (See also Zhang et al., NucleicAcid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res.25:3389-402 (1997), which are incorporated by reference herein).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information internet web site. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.(1990), supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9(1992), which is incorporated by reference herein) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci.USA 90:5873-77 (1993), which is incorporated by reference herein). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance. For example, a nucleic acid is considered similar to areference sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about0.1, more typically less than about 0.01, and most typically less thanabout 0.001.

In another embodiment, targeting agents other than the RVG peptide areencompassed for use in the present invention. Examples of such targetingagents include, but are not limited to other molecules capable ofdelivering attached cargo across the BBB. Such BBB targeting agents canbe any of the known targeting moieties that undergo receptor mediatedtransport across the BBB via endogenous peptide receptor transportsystems localized in the brain capillary endothelial plasma membrane,which forms the BBB in vivo. In some embodiments, targeting agentsuseful in the methods of the present invention are, for example but notlimited to insulin, transferrin, insulin-like growth factor (IGF),leptin, low density lipoprotein (LDL), and the corresponding peptides,peptide fragments, peptidomimetics or derivatives thereof, as well asmonoclonal antibodies that mimic these endogenous peptides. In someembodiment, a targeting agent is a avimer of fragments, for example butnot limited to the binding domains of insulin, transferrin, insulin-likegrowth factor (IGF), leptin or low density lipoprotein (LDL) proteins,thus enabling multi-receptor targeting of receptors expressed on thesurface of cells of the BBB.

Without being bound by theory, peptidomimetic monoclonal antibodies arealso useful as targeting agents in the methods of the present invention.Such antibodies bind to exofacial epitopes on the BBB receptor, removedfrom the binding site of the endogenous peptide ligand, and “piggyback”across the BBB via the endogenous peptide receptor-mediated transcytosissystem. Peptidomimetic monoclonal antibodies are species specific. Forexample, the OX26 murine monoclonal antibody to the rat transferrinreceptor is used for drug delivery to the rat brain (Pardridge et al.1991. J Pharmacol Exp Ther 256:66-70); however, variants or derivativesof transferring targeting agents are preferred in humans (Lee et al.2000. J Pharmacol. Exp Ther 292: 1048-1052). Monoclonal antibodies tothe human insulin receptor (HIR) are also useful for delivering thepharmaceutical composition to the human brain. In some embodiments,“humanized” monoclonal antibodies are used. Exemplary, humanizedmonoclonal antibodies to the human insulin receptor that areparticularly well-suited for use in the present invention are describedin detail in U.S. Patent App. No. 2004/0101904, the contents of whichapplication are hereby specifically incorporated by reference. Othertargeting agents useful in the methods as disclosed herein are, forexample the rat 8D3 or rat RI7-217 monoclonal antibody to the mousetransferrin receptor for drug delivery to mouse brain (Lee et al. 2000.J Pharmacol Exp Ther 292: 1048-1052), or murine, chimeric or humanizedantibodies to the human or animal transferrin receptor, the human oranimal leptin receptor, the human or animal IGF receptor, the human oranimal LDL receptor, the human or animal acetylated LDL receptor.

The term “targeting agent” or “targeting moiety” refers to an agent thathomes in on or selectively targets or preferentially associates or bindsto a particular tissue, cell type, receptor, or other molecule orparticle f interest. In the methods of the present invention, thetargeting agent promotes transport or preferential localization of aneffector agent to the target of interest, i.e., neuronal cells. Onetargeting agent of the present invention comprises an amino acidsequence derived from the rabies virus glycoprotein (RVG) that iseffective in binding to cells expressing the a subunit of theacetylcholine receptor, including, for example, brain cells, spinal cordcells, neuronal cells, glia cells and endothelial cells comprising theBBB.

As used herein, the term “target cells” is used herein to refer to cellswhich sit entirely within BBB-protected CNS tissue. The term “targetcells” as used herein also refers to cells expressing the a subunit ofthe acetylcholine receptor. In one embodiment, the target cells expressa type 1 and/or a type 7 acetylcholine receptor. An RVG peptide asdisclosed herein binds to the α-subunit of the acetylcholine receptor.Accordingly, an RVG peptide as disclosed herein is useful as a targetingmoiety for the selective targeting of cells expressing the a subunit ofthe acetylcholine receptor. Cells expressing the a subunit of theacetylcholine receptor include, for example, neurons, glial cells andendothelial cells comprising the blood brain barrier. Target cells ofthe present invention also include, cells whose endogenous milieu isseparated by the BBB, for example, cells in the central nervous system,e.g., brain cells, spinal cord cells, glial cells and other cellssupporting neurons, for e.g. astrocytes or “nursing cells” and cells ofthe central nervous system. In some embodiments, the target cells can beany cell expressing the a subunit of acetylcholine receptor or ahomologue thereof, such as for example but not limited to neuronal cellsin a subject (i.e. in vivo), neuronal cells ex vivo or cultured neuronalcells (i.e. in vitro) such as, for example as primary neuronal culturedcells. In some embodiments, the target cells are neuronal precursor orneuronal progenitor cells, such as neuronal progenitor stem cells thatexpress the a subunit of the acetylcholine receptor or a homologuethereof.

The term “glial cells” or “glia” (also called neuroglial cells), whichare used interchangeably herein refers to various types of cells whichcannot receive or transmit nerve signals, and which instead support andserve the neurons located inside the BBB. These glial cells performvarious activities that can be regarded as supporting, housekeeping, and“nursing” functions within the CNS. Glial cells are divided into variouscategories, including oligodendroglia cells, astrocytes, ependymalcells, and microglia cells and are commonly known by persons of ordinaryskill in the art.

The term “selectively target” as used herein refers to the ability of atargeting agent to home in on or bind to a target cell with a greateraffinity than to non-target cells. For example, about 10%, about 20%,about 30%, about 40%, preferably about 50%, more preferably about 60%,more preferably about 70%, still more preferably about 80%, still morepreferably about 90%, still more preferably about 100% or greateraffinity for the target cell relative to non-target cells.

The term “amino acid” is used in its broadest sense, and includesnaturally occurring amino acids as well as non-naturally occurring aminoacids, including amino acid analogs and derivatives. For example,homo-phenylalanine, citrulline, and norleucine are considered aminoacids for the purposes of the invention. “Amino acids” also includesamino residues such as proline and hydroxyproline. The side chains canbe either the (R) or (S) configuration. If non-naturally occurring sidechains are used, non-amino acid substituents can be used.

The term “peptide” as used herein, refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, peptides contain at least two aminoacid residues and are less than about 50 amino acids in length.

The terms “peptide” “polypeptide” and “protein” are used interchangeablyto refer to a polymer of amino acid residues, and are not limited to aminimum length. Thus, peptides, oligopeptides, dimers, multimers, andthe like, whether produced biologically, recombinantly, or syntheticallyand whether composed of naturally occurring or non-naturally occurringamino acids, are included within this definition. Both full-lengthproteins and fragments thereof are encompassed by the definition. Theterms also include co-translational (e.g., signal peptide cleavage) andpost-translational modifications of the polypeptide, such as, forexample, disulfide-bond formation, glycosylation, acetylation,phosphorylation, proteolytic cleavage (e.g., cleavage by furins ormetalloproteases), and the like. Furthermore, for purposes of thepresent invention, a “polypeptide” refers to a protein that includesmodifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart), to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods.

The term “recombinant” as used herein to describe a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic,and/or synthetic origin, which, by virtue of its origin or manipulation,is not associated with all or a portion of the polynucleotide with whichit is associated in nature. The term recombinant as used with respect toa protein or polypeptide, means a polypeptide produced by expression ofa recombinant polynucleotide. The term recombinant as used with respectto a host cell means a host cell into which a recombinant polynucleotidehas been introduced. Recombinant is also used herein to refer to, withreference to material (e.g., a cell, a nucleic acid, a protein, or avector) that the material has been modified by the introduction of aheterologous material (e.g., a cell, a nucleic acid, a protein, or avector).

The term “polymer” as used herein, refers to a linear chain of two ormore identical or non-identical subunits joined by covalent bonds. Apeptide is an example of a polymer that can be composed of identical ornon-identical amino acid subunits that are joined by peptide linkages.

As used herein, the term “gene” refers to a nucleic acid comprising anopen reading frame encoding a polypeptide, including both exon and(optionally) intron sequences. A “gene” refers to coding sequence of agene product, as well as non-coding regions of the gene product,including 5′UTR and 3′UTR regions, introns and the promoter of the geneproduct. These definitions generally refer to a single-strandedmolecule, but in specific embodiments will also encompass an additionalstrand that is partially, substantially or fully complementary to thesingle-stranded molecule. Thus, a nucleic acid can encompass adouble-stranded molecule or a double-stranded molecule that comprisesone or more complementary strand(s) or “complement(s)” of a particularsequence comprising a molecule. As used herein, a single strandednucleic acid can be denoted by the prefix “ss”, a double strandednucleic acid by the prefix “ds”, and a triple stranded nucleic acid bythe prefix “ts.” The term “gene” refers to the segment of DNA involvedin producing a polypeptide chain, it includes regions preceding andfollowing the coding region as well as intervening sequences (introns)between individual coding segments (exons). A “promoter” is a region ofa nucleic acid sequence at which initiation and rate of transcriptionare controlled. It can contain elements at which regulatory proteins andmolecules can bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription of a nucleic acidsequence. The term “enhancer” refers to a cis-acting regulatory sequenceinvolved in the transcriptional activation of a nucleic acid sequence.An enhancer can function in either orientation and can be upstream ordownstream of the promoter. As used herein, the term “gene product(s)”is used to refer to include RNA transcribed from a gene, or apolypeptide encoded by a gene or translated from RNA.

The term “protein” as used herein, refers to a compound that is composedof linearly arranged amino acids linked by peptide bonds, but incontrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the peptides (or other components of the composition, withexception for protease recognition sequences) is desirable in certainsituations. D-amino acid-containing peptides exhibit increased stabilityin vitro or in vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides.

In yet a further embodiment, the peptides are retro-inverso peptides. A“retro-inverso peptide” refers to a peptide with a reversal of thedirection of the peptide bond on at least one position, i.e., a reversalof the amino- and carboxy-termini with respect to the side chain of theamino acid. Thus, a retro-inverso analogue has reversed termini andreversed direction of peptide bonds while approximately maintaining thetopology of the side chains as in the native peptide sequence. Theretro-inverso peptide can contain L-amino acids or D-amino acids, or amixture of L-amino acids and D-amino acids, up to all of the amino acidsbeing the D-isomer. Partial retro-inverso peptide analogues arepolypeptides in which only part of the sequence is reversed and replacedwith enantiomeric amino acid residues. Since the retro-inverted portionof such an analogue has reversed amino and carboxyl termini, the aminoacid residues flanking the retro-inverted portion are replaced byside-chain-analogous α-substituted geminal-diaminomethanes andmalonates, respectively. Retro-inverso forms of cell penetratingpeptides have been found to work as efficiently in translocating acrossa membrane as the natural forms. Synthesis of retro-inverso peptideanalogues are described in Bonelli, F. et al., Int J Pept Protein Res.24(6):553-6 (1984); Verdini, A and Viscomi, G. C., J. Chem. Soc. PerkinTrans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which areincorporated herein in their entirety by reference. Processes for thesolid-phase synthesis of partial retro-inverso peptide analogues havebeen described (EP 97994-B) which is also incorporated herein in itsentirety by reference.

In one embodiment, the targeted delivery composition includes polymers,for example peptides such as an RVG peptide and a peptide carrierparticle, comprised of D- or L-amino acid residues. Use of naturallyoccurring L-amino acid residues in the transport polymers has theadvantage that break-down products should be relatively non-toxic to thecell or organism.

In some embodiments, the peptide carrier particle comprises arginineamino acid subunits, for example α-amino-β-guanidi-novaleric acid andα-amino-ε-amidinohexanoic acid (isosteric amidino analog). In someembodiments, the guanidinium group in arginine has a pKa of about 12.5.More generally, in some embodiments each polymer subunit of a peptidecarrier particle contains a highly basic sidechain moiety which (i) hasa pKa of greater than 11, more preferably 12.5 or greater, and (ii)contains, in its protonated state, at least two geminal amino groups(NH₂) which share a resonance-stabilized positive charge, which givesthe moiety a bidentate character. Other amino acids, such asα-amino-β-guanidinopropionic acid, α-amino-γ-guanidinobutyric acid, orα-amino-ε-guanidinocaproic acid can also be used (containing 2, 3 or 5linker atoms, respectively, between the backbone chain and the centralguanidinium carbon).

In alternative embodiments, the peptides as disclosed herein, forexample an RVG peptide and/or peptide carrier particles can compriseD-amino acids. Compositions containing exclusively D-amino acids havethe advantage of decreased enzymatic degradation. However, they can alsoremain largely intact within the target cell. Such stability isgenerally not problematic if the agent being delivered to the cell isbiologically active when a peptide carrier particle is still attached.For agents that are inactive when conjugated with a peptide carrierparticle, a linker that is cleavable and can be cleaved by a suitablemechanism in a target cell (e.g., by enzyme- or solvent-mediatedcleavage within a cell) can be included to promote release of the agentfrom the peptide carrier particle in the target cell.

Carrier Particles

The carrier particles of the effector agents include any carrierparticle modifiable by attachment of a targeting agent known to theskilled artisan. Carrier particles include but are not limited toliposomal or polymeric nanoparticles such as liposomes, proteins, andnon-protein polymers. Carrier particles can be selected according totheir ability to transport the effector agent of choice and the abilityto covalently attach the targeting agent to the carrier particle.

In some embodiments, carrier particles include colloidal dispersionsystems, which include, but are not limited to, macromolecule complexes,nanocapsules, microspheres, beads and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, liposomes andlipid:oligonucleotide complexes of uncharacterized structure. In someembodiments, the carrier particle comprises a plurality of liposomes.Liposomes are microscopic spheres having an aqueous core surrounded byone or more outer layers made up of lipids arranged in a bilayerconfiguration (see, generally, Chonn et al., Current Op. Biotech. 1995,6, 698-708). Other carrier particles are cellular uptake ormembrane-disruption moieties, for example polyamines, e.g. spermidine orspermine groups, or polylysines; lipids and lipophilic groups; polymyxinor polymyxin-derived peptides; octapeptin; membrane pore-formingpeptides; ionophores; protamine; aminoglycosides; polyenes; and thelike. Other potentially useful functional groups include intercalatingagents; radical generators; alkylating agents; detectable labels;chelators; or the like.

One can use other carrier particles, for example lipid particle orvesicle, such as a liposome or microcrystal, which may be suitable forparenteral administration. The particles may be of any suitablestructure, such as unilamellar or plurilamellar, so long as theantisense oligonucleotide is contained therein. Positively chargedlipids such asN-[I-(2,3dioleoyloxi)propyll-N,N,N-trimethyl-anunoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. See, e.g., U.S. Pat.Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and4,921,757 which are incorporated herein by reference. Other non-toxiclipid based vehicle components may likewise be utilized to facilitateuptake of the antisense compound by the cell.

In some embodiments, a carrier particle is a liposome. The outer surfaceof the liposomes can be modified with a long-circulating agent, e.g.,PEG, e.g., hyaluronic acid (HA). The liposomes can be modified with acryoprotectant, e.g., a sugar, such as trehalose, sucrose, mannose orglucose, e.g., HA. In one embodiment, a liposome is coated with HA. HAacts as both a long-circulating agent and a cryoprotectant. The liposomeis modified by attachment of the targeting moiety. In anotherembodiment, the targeting moiety is covalently attached to HA, which isbound to the liposome surface. Alternatively, the carrier particle is amicelle. Alternatively, the micelle is modified with a cryoprotectant,e.g., HA, PEG.

Liposomes useful in the methods and compositions as disclosed herein canbe produced from combinations of lipid materials well known androutinely utilized in the art to produce liposomes. Lipids can includerelatively rigid varieties, such as sphingomyelin, or fluid types, suchas phospholipids having unsaturated acyl chains. “Phospholipid” refersto any one phospholipid or combination of phospholipids capable offorming liposomes. Phosphatidylcholines (PC), including those obtainedfrom egg, soy beans or other plant sources or those that are partiallyor wholly synthetic, or of variable lipid chain length and unsaturationare suitable for use in the present invention. Synthetic, semisyntheticand natural product phosphatidylcholines including, but not limited to,distearoylphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), eggphosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine(HEPC), dipalmitoylphosphatidylcholine (DPPC) anddimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholinesfor use in this invention. All of these phospholipids are commerciallyavailable. Further, phosphatidylglycerols (PG) and phosphatic acid (PA)are also suitable phospholipids for use in the present invention andinclude, but are not limited to, dimyristoylphosphatidylglycerol (DMPG),dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol(DPPG), distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidicacid (DMPA), distearoylphosphatidic acid (DSPA), dilaurylphosphatidicacid (DLPA), and dipalmitoylphosphatidic acid (DPPA).Distearoylphosphatidylglycerol (DSPG) is the preferred negativelycharged lipid when used in formulations. Other suitable phospholipidsinclude phosphatidylethanolamines, phosphatidylinositols,sphingomyelins, and phosphatidic acids containing lauric, myristic,stearoyl, and palmitic acid chains. For the purpose of stabilizing thelipid membrane, it is preferred to add an additional lipid component,such as cholesterol. Preferred lipids for producing liposomes accordingto the invention include phosphatidylethanolamine (PE) andphosphatidylcholine (PC) in further combination with cholesterol (CH).According to one embodiment of the invention, a combination of lipidsand cholesterol for producing the liposomes of the invention comprise aPE:PC:Chol molar ratio of 3:1:1. Further, incorporation of polyethyleneglycol (PEG) containing phospholipids is also contemplated by thepresent invention.

Liposomes useful in the methods and compositions as disclosed herein canbe obtained by any method known to the skilled artisan. For example, theliposome preparation of the present invention can be produced by reversephase evaporation (REV) method (see U.S. Pat. No. 4,235,871), infusionprocedures, or detergent dilution. A review of these and other methodsfor producing liposomes can be found in the text Liposomes, Marc Ostro,ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. See also Szoka Jr.et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467). A method for formingULVs is described in Cullis et al., PCT Publication No. 87/00238, Jan.16, 1986, entitled “Extrusion Technique for Producing UnilamellarVesicles”. Multilamellar liposomes (MLV) can be prepared by thelipid-film method, wherein the lipids are dissolved in achloroform-methanol solution (3:1, vol/vol), evaporated to dryness underreduced pressure and hydrated by a swelling solution. Then, the solutionis subjected to extensive agitation and incubation, e.g., 2 hour, e.g.,at 37° C. After incubation, unilamellar liposomes (ULV) are obtained byextrusion. The extrusion step modifies liposomes by reducing the size ofthe liposomes to a preferred average diameter. Alternatively, liposomesof the desired size can be selected using techniques such as filtrationor other size selection techniques. While the size-selected liposomes ofthe invention should have an average diameter of less than about 300 nm,it is preferred that they are selected to have an average diameter ofless than about 200 nm with an average diameter of less than about 100nm being particularly preferred. When the liposome of the presentinvention is a unilamellar liposome, it preferably is selected to havean average diameter of less than about 200 nm. The most preferredunilamellar liposomes of the invention have an average diameter of lessthan about 100 nm. It is understood, however, that multivesicularliposomes of the invention derived from smaller unilamellar liposomeswill generally be larger and can have an average diameter of about lessthan 1000 nm. Preferred multivesicular liposomes of the invention havean average diameter of less than about 800 nm, and less than about 500nm while most preferred multivesicular liposomes of the invention havean average diameter of less than about 300 nm.

A method for coating the liposomes or other polymeric nanoparticles withtargeting agents, such as an RVG peptide comprising SEQ ID NO:13 orvariants, derivatives or fragments thereof are disclosed in U.S.Provisional Application No. 60/794,361 filed Apr. 24, 2006, andInternational Patent Application: PCT/US07/10075 filed Apr. 24, 2007with are incorporated in their entirety herein by reference.

In some embodiments, the outer surface of the liposomes can be furthermodified with a long-circulating agent. The modification of theliposomes with a hydrophilic polymer as the long-circulating agent isknown to enable to prolong the half-life of the liposomes in the blood.Examples of the hydrophilic polymer include polyethylene glycol,polymethylethylene glycol, polyhydroxypropylene glycol, polypropyleneglycol, polymethylpropylene glycol and polyhydroxypropylene oxide. Inone embodiment, a hydrophilic polymer is polyethylene glycol (PEG).Glycosaminoglycans, e.g., hyaluronic acid, can also be used aslong-circulating agents.

In some embodiments, the targeting agent, such as an RVG peptidecomprising SEQ ID NO:13 or a derivative, variant or fragment thereof isconjugated to a cryoprotectant present on the liposome, e.g., HA.Crosslinking reagents include glutaraldehyde (GAD), bifunctional oxirane(OXR), ethylene glycol diglycidyl ether (EGDE), N-hydroxysuccinimide(NETS), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). As is known to theskilled artisan, any crosslinking chemistry can be used, including, butnot limited to, thioether, thioester, malimide and thiol,amine-carboxyl, amine-amine, and others listed in organic chemistrymanuals, such as, Elements of Organic Chemistry, Isaak and HenryZimmerman Macmillan Publishing Co., Inc. 866 Third Avenue, New York,N.Y. 10022. Through the complex chemistry of crosslinking, linkage ofthe amine residues of the recognizing substance and liposomes isestablished.

In some embodiments, after the targeting agent is conjugated orcovalently attached to the lipid particle by way of covalent linkage tothe cryoprotectant, or by way of covalent linkage to another targetingagent covalently linked to the cryoprotectant, the lipid particle may belyophilized. The lipid particle may remain lyophilized prior torehydration, or prior to rehydration and encapsulation of the agent ofinterest, for extended periods of time. In one embodiment, the lipidparticle remains lyophilized for about 1 month, about 2 months, about 3months, about 6 months, about 9 months, about 12 months, about 18months, about 2 years or more prior to rehydration.

In another embodiment, the carrier particle is a cyclodextrin-basednanoparticle. Polycation formulated nanoparticles have been used fordrug delivery into the brain as well as for systemic delivery of siRNA[114, 115]. A unique cyclodextrin-based nanoparticle technology has beendeveloped for targeted gene delivery in vivo [116-123]. This deliverysystem consists of two components. The first component is a biologicallynon-toxic cyclodextrin-containing polycation (CDP). CDPs self-assemblewith siRNA to form colloidal particles about 50 nm in diameter andprotects si/shRNA against degradation in body fluids. Moreover, the CDPhas been engineered to contain imidazole groups at their termini toassist in the intracellular trafficking and release of the nucleic acid[123]. CDP also enables assembly with the second component. The secondcomponent is an adamantane-terminated polyethylene glycol (PEG) modifierfor stabilizing the particles in order to minimize interactions withplasma and to attach cell surface targeting molecules such astransferrin or RVG peptides. Thus, the advantages of this deliverysystem are: 1) since the CDP protects the siRNA from degradation,chemical modification of the nucleic acid is unnecessary, 2) thecolloidal particles do not aggregate and have extended life inbiological fluids because of the surface decoration with PEG that occursvia inclusion complex formation between the terminal adamantane and thecyclodextrins [123], 3) cell type-specific targeted delivery is possiblebecause some of the PEG chains contain targeting ligands, 4) it does notinduce an immune response [116, 119], and 5) in vivo delivery does notproduce an interferon response even when a siRNA is used that contains amotif known to be immunostimulatory when delivered in vivo with lipids[116].

In another embodiment, the carrier particle is a cationic peptide, e.g.,protamine. See, for example, WO 06/023491, which is specificallyincorporated herein in its entirety by reference.

The glycosaminoglycan carrier particles disclosed in U.S. Patent Appl.No. 20040241248 and the glycoprotein carrier particles in WO 06/017195,which are incorporated herein in their entirety by reference, are usefulin the methods of the present invention. Similar naturally occurringpolymer-type carriers known to the skilled artisan are also useful inthe methods of the present invention.

Soluble non-protein polymers are useful as carrier particles. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylrnethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palitoyl residues. Furthermore, the therapeutic agentscan be coupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathicblock copolymers of hydrogels. The therapeutic agents can also beaffixed to rigid polymers and other structures such as fullerenes orBuckeyballs.

The carrier particle can be conjugated to the targeting agent, forexample RVG peptide or variant thereof. The conjugation can be anon-covalent or covalent interaction, for example, by means of chemicalcrosslinkage or conjugation.

As used herein, the term “conjugate” or “conjugation” refers to theattachment of two or more entities to form one entity. For example, themethods of the present invention provide conjugation of a targetingagent (for example an RVG peptide or variant or fragment or derivative)joined with another entity, for example a carrier particle, for examplea liposome or cell penetrating agent, for e.g. a cell penetratingpeptide. The attachment can be by means of linkers, chemicalmodification, peptide linkers, chemical linkers, covalent ornon-covalent bonds, or protein fusion or by any means known to oneskilled in the art. The joining can be permanent or reversible. In someembodiments, several linkers can be included in order to take advantageof desired properties of each linker and each protein in the conjugate.Flexible linkers and linkers that increase the solubility of theconjugates are contemplated for use alone or with other linkers asdisclosed herein. Peptide linkers can be linked by expressing DNAencoding the linker to one or more proteins in the conjugate. Linkerscan be acid cleavable, photocleavable and heat sensitive linkers.Methods for conjugation are well known by persons skilled in the art andare encompassed for use in the present invention.

According to the present invention, the targeting agent, for example anRVG peptide, can be linked to the carrier particle entity via anysuitable means, as known in the art, see for example U.S. Pat. Nos.4,625,014, 5,057,301 and 5, 514,363, which are incorporated herein intheir entirety by reference. For example, the agent to be transportedcan be covalently conjugated to the transporting entity, either directlyor through one or more linkers. In one embodiment, the transportingentity of the present invention is conjugated directly to an agent to betransported. In another embodiment, the transporting entity of thepresent invention is conjugated to an agent to be transported via alinker, e.g. a transport enhancing linker.

A large variety of methods for conjugation of targeting agents withcarrier particles or diagnostic moieties are known in the art. Suchmethods are e.g. described by Hermanson (1996, Bioconjugate Techniques,Academic Press), in U.S. Pat. No. 6,180,084 and U.S. Pat. No. 6,264,914which are incorporated herein in their entirety by reference and includee.g. methods used to link haptens to carriers proteins as routinely usedin applied immunology (see Harlow and Lane, 1988, “Antibodies: Alaboratory manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). It is recognized that, in some cases, a targeting agentor carrier particle can lose efficacy or functionality upon conjugationdepending, e.g., on the conjugation procedure or the chemical grouputilised therein. However, given the large variety of methods forconjugation the skilled person is able to find a conjugation method thatdoes not or least affects the efficacy or functionality of the entitiesto be conjugated.

Suitable methods for conjugation of a targeting agent with carrierparticle include e.g. carbodimide conjugation (Bauminger and Wilchek,1980, Meth. Enzymol. 70: 151-159). Alternatively, a moiety can becoupled to a targeting agent as described by Nagy et al., Proc. Natl.Acad. Sci. USA 93:7269-7273 (1996), and Nagy et al., Proc. Natl. Acad.Sci. USA 95:1794-1799 (1998), each of which are incorporated herein byreference. Another method for conjugating one can use is, for examplesodium periodate oxidation followed by reductive alkylation ofappropriate reactants and glutaraldehyde crosslinking.

One can use a variety of different linkers to conjugate the targetingagent, for example RVG peptide as described herein to a carrierparticle, for example but not limited to aminocaproic horse radishperoxidase (HRP) or a heterobiofunctional cross-linker, e.g. carbonylreactive and sulfhydryl-reactive cross-linker. Heterobiofunctional crosslinking reagents usually contain two reactive groups that can be coupledto two different function targets on proteins and other macromoleculesin a two or three-step process, which can limit the degree ofpolymerization often associated with using homobiofunctionalcross-linkers. Such multistep protocols can offer a great control ofconjugate size and the molar ratio of components.

The term “linker” refers to any means to join two or more entities, forexample a peptide with another peptide, or a liposome. A linker can be acovalent linker or a non-covalent linker. Examples of covalent linkersinclude covalent bonds or a linker moiety covalently attached to one ormore of the proteins to be linked. The linker can also be a non-covalentbond, e.g. an organometallic bond through a metal center such asplatinum atom. For covalent linkages, various functionalities can beused, such as amide groups, including carbonic acid derivatives, ethers,esters, including organic and inorganic esters, amino, urethane, ureaand the like. To provide for linking, the effector molecule and/or theprobe can be modified by oxidation, hydroxylation, substitution,reduction etc. to provide a site for coupling. It will be appreciatedthat modification which do not significantly decrease the function ofthe target agent, for example RVG peptide and/or the carrier particleare preferred.

In some embodiments where the carrier particle is a liposome orpolymeric nanoparticle, the effector agent and/or targeting agent, suchas an RVG peptide is captured within a liposomes or polymericnanoparticle or immunoliposomes. For example, a suspension of RVGpeptide or variant or fragment thereof and/or effector agent can beencapsulated in micelles to form liposomes by conventional methods (U.S.Pat. No. 5,043,164, U.S. Pat. No. 4,957, 735, I5 U.S. Pat. No.4,925,661; Connor and Huang, (1985) J. Cell Biol. 101: 581; Lasic D. D.(1992) Nature 355: 279; Novel Drug Delivery (eds. Prescott and Nimmo,Wiley, New York-, 1989); Reddy et al. (1992) J. Immunol. 148:1585),which are incorporated herein in their entirety by reference. Liposomescomprising targeting agent that binds specifically to neurons (e.g.,neurons expressing acetylcholine receptor (AchR)) or cells of the bloodbrain barrier can be used to target the agents to those cells. The terms“encapsulation” and “entrapped,” as used herein, refer to theincorporation of an agent in a lipid particle. The agent is present inthe aqueous interior of the lipid particle. In one embodiment, a portionof the encapsulated agent takes the form of a precipitated salt in theinterior of the liposome. The agent may also self precipitate in theinterior of the liposome.

In another embodiment where the effector agent is a nucleic acid, e.g.,DNA, RNA, siRNA, plasmid DNA, short-hairpin RNA, small temporal RNA(stRNA), microRNA (miRNA), RNA mimetics, or heterochromatic siRNA, thenucleic acid effector agent has a charged backbone that preventsefficient encapsulation in the lipid particle. Accordingly, the nucleicacid effector agent of interest may be condensed with a cationicpolymer, e.g., PEI, polyamine spermidine, and spermine, or cationicpeptide, e.g., protamine and polylysine, prior to encapsulation in thelipid particle. In some embodiments, the effector agent is not condensedwith a cationic polymer.

In some embodiments, an effector agent is encapsulated in the lipidparticle or other polymeric nanoparticle in the following manner. Thelipid particle or polymeric nanoparticle, in which can additionallycomprise a cryoprotectant and/or a targeting agent is providedlyophilized. The effector agent is in an aqueous solution. The effectoragent in aqueous solution is utilized to rehydrate the lyophilized lipidparticle or nanoparticle. Thus, the effector agent is encapsulated inthe rehydrated lipid particle or polymeric nanoparticle. For example butnot limited to, the cDNA for the glial cell line derived neurogrowthfactor (GDNF) may be targeted to the dopamine cells at the substantianigra in Parkinson's disease patients.

In some embodiments, two or more effector agents can be delivered by thelipid particle or polymeric nanoparticles by the methods as disclosedherein. In such embodiments, one agent can be hydrophobic and the otherhydrophilic. The hydrophobic agent can be added to the lipid particleduring formation of the lipid particle. The hydrophobic agent associateswith the lipid portion of the lipid particle. The hydrophilic agent isadded in the aqueous solution rehydrating the lyophilized lipidparticle. An exemplary embodiment of two agent delivery is describedbelow, wherein a condensed siRNA is encapsulated in a liposome andwherein a drug that is poorly soluble in aqueous solution is associatedwith the lipid portion of the lipid particle. As used herein, “poorlysoluble in aqueous solution” refers to a composition that is less that10% soluble in water.

Any suitable lipid: pharmaceutical agent ratio that is efficacious iscontemplated by this invention. Preferred lipid: pharmaceutical agentmolar ratios include about 2:1 to about 30:1, about 5:1 to about 100:1,about 10:1 to about 40:1, about 15:1 to about 25:1.

The preferred loading efficiency of therapeutic or pharmaceutical agentis a percent encapsulated pharmaceutical agent of about 50%, about 60%,about 70% or greater. In one embodiment, the loading efficiency for ahydrophilic agent is a range from 50-100%. The preferred loadingefficiency of pharmaceutical agent associated with the lipid portion ofthe lipid particle, e.g., a pharmaceutical agent poorly soluble inaqueous solution, is a percent loaded pharmaceutical agent of about 50%,about 60%, about 70%, about 80%, about 90%, about 100%. In oneembodiment, the loading efficiency for a hydrophobic agent in the lipidlayer is a range from 80-100%.

In one aspect of the method, the liposome or polymeric nanoparticle isdetectably labeled with a label selected from the group including aradioactive label, a fluorescent label, a non-fluorescent label, a dye,or a compound which enhances magnetic resonance imaging (MRI). In oneembodiment, the liposome product is detected by acoustic reflectivity.The label may be attached to the exterior of the liposome or may beencapsulated in the interior of the liposome.

In some embodiments, and in the event that the carrier particle is apeptide or protein, and the targeting agent is also a peptide orantibody, or contains amino acids as part of its structure, thetargeting agent (for example an RVG peptide) can be fused either inframe or out of frame with the carrier particle to form a fusionprotein. In general, the targeting agent (i.e. an RVG peptide) andcarrier particle can be fused directly or via one or more amino acidlinkers. Any suitable amino acid linkers can be used to modify thestability, conformation, charge, or other structure features of theresulting fusion protein in order to facilitate its transport to targetcells. In some embodiments, fusion proteins can also be formed from thecarrier particle and effector agent, where both the carrier particle andeffector agents are proteins or contain amino acids as part of theirstructure, and preferably the activity of the effector agent is notcompromised by being fused with the carrier particle.

The term “fusion protein” refers to a recombinant protein of two or morefused proteins. Fusion proteins can be produced, for example, by anucleic acid sequence encoding one protein joined to the nucleic acidencoding another protein such that they constitute a single open-readingframe that can be translated in the cells into a single polypeptideharboring all the intended proteins. The order of arrangement of theproteins can vary. As a non-limiting example, the nucleic acid sequenceencoding an RVG peptide can be fused to either the 5′ or the 3′ end ofthe nucleic acid sequence encoding a carrier particle. In this manner,on expression of the nucleic acid construct, the RVG peptide isfunctionally expressed and fused to the N-terminal or C-terminal end ofthe carrier protein. In certain embodiments, the carrier peptide can bemodified such that the carrier protein function (i.e. ability toassociate with the effector agent) remains unaffected by fusion to theRVG peptide and vice versa, the RVG peptide can be modified, for exampleRVG peptide variants can be used so that the RVG peptide retains theability to penetrate or pass through the BBB even when fused withanother protein, for example the carrier particle.

The targeted delivery composition can also be prepared via recombinantexpression through bacterial or eukaryotic expression systems of aprotein-based carrier particle, for example HIV-TAT (SEQ ID NO:15) or afragment thereof, protamine, and an RVG peptide. Bacterial andeukaryotic expression systems are well known and used routinely by thoseskilled in the art. Accordingly, in one embodiment a targeted deliverycomposition comprises an RVG peptide or variant or fragment orderivative thereof, and a polymeric arginine residue of a varyinglength, such as 9R or 11R as disclosed herein. An example of one suchtargeted delivery composition is RVG-11dR:YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRRRR (SEQ ID NO: 14). Anotherexample of one such targeted delivery composition is RVG-9dR:YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR (SEQ ID NO: 34). Anotherexample of such a targeted delivery composition comprise an RVG peptideand a protamine peptide: YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 16). In another embodiment, a targeted deliverycomposition comprises an RVG peptide and TAT:YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGYGRKKRRQRRRKKRK (SEQ ID NO: 15). In someembodiments, SEQ ID NO:14 and SEQ ID NO:34 can gave arginine (R)sequentially deleted or added from the C-terminal end, for exampledeleted by 1, 2, 3, 4, 5, or 6 C-terminal R residues.

In some embodiments, the carrier particle also comprises a cellpenetrating agent. A cell penetrating agent can also be covalentlyattached to a carrier particle, e.g., a liposomal carrier particle. Insome embodiments, the composition of the present invention includes acell penetrating agent and a separate carrier particle. For example, acell penetrating agent can be attached to a liposomal carrier particle.

Any peptide or fragment thereof, which is capable of crossing abiological membrane, either in vivo or in vitro, is encompassed for usein the methods and compositions of the present invention. Such peptidesthat are capable of crossing membranes are termed “cell penetratingagents”. “Cell penetrating agents” or “translocation agents “compriseagents that facilitate delivery of an associated compound of interest orcargo across a cell membrane. It is known that certain peptides have theability to penetrate a lipid bilayer (e.g., cell membranes) andtranslocate an attached cargo across the cell membrane. This is referredto herein as “translocation activity”. Without being bound by theory,these membrane penetrating peptides appear to enter the cell, in part,via non-endocytic mechanisms, as indicated by the ability of the cellpenetrating peptides to enter the cell at low temperatures (e.g., 4° C.)that would normally inhibit endocytic, receptor-based, internalizationpathways.

Generally, the cell penetrating agents are capable of facilitatingtransfer of a cargo or compound such as an effector agent across a lipidbilayer in a non-selective manner because entry into the cell does notappear to occur by receptor-mediated endocytic pathway. Consequently,the cell penetrating agent is capable of translocating cargoesnon-selectively into a variety of cell types. In some embodiments, tocontrol delivery of the compositions into cell types, the compositionscan further comprise a cell penetrating peptide inhibitor or aninhibitor of cell penetrating peptide. Modification of the inhibitorresults in release of the inhibitory effect and formation of an activecell penetrating composition.

In some embodiments, the cell penetrating agent is a cell penetratingpeptide. Methods to synthesize such peptides are well known to one ofordinary skill in the art. For example, several cell penetratingpeptides have been identified which can be used as carrier peptides inthe methods of the invention for transporting RNA interfering agentsacross biological membranes. These peptides include, for example, thehomeodomain of antennapedia, a Drosophila transcription factor (Wang etal., (1995) PNAS USA., 92, 3318-3322); a fragment representing thehydrophobic region of the signal sequence of Kaposi fibroblast growthfactor with or without NLS domain (Antopolsky et al. (1999) Bioconj.Chem., 10, 598-606); a signal peptide sequence of caiman crocodylusIg(5) light chain (Chaloin et al. (1997) Biochem. Biophys. Res. Comm.,243, 601-608); a fusion sequence of HIV envelope glycoprotein gp4114,(Morris et al. (1997) Nucleic Acids Res., 25, 2730-2736); a transportanA-achimeric 27-mer consisting of N-terminal fragment of neuropeptidegalanine and membrane interacting wasp venom peptide mastoporan(Lindgren et al., (2000), Bioconjugate Chem., 11, 619-626); a peptidederived from influenza virus hemagglutinin envelop glycoprotein(Bongartz et al., 1994, Nucleic Acids Res., 22, 468 1 4688); RGDpeptide; and a peptide derived from the human immunodeficiency virustype-1 (“HIV-1”). Purified HIV-1 TAT protein is taken up from thesurrounding medium by human cells growing in culture (Frankel and Pabo,(1988) Cell, 55, pp. 1189-93). TAT protein trans-activates certain HIVgenes and is essential for viral replication. The full-length HIV-1 TATprotein has 86 amino acid residues. The HIV tat gene has two exons. TATamino acids 1-72 are encoded by exon 1, and amino acids 73-86 areencoded by exon 2. The full-length TAT protein is characterized by abasic region which contains two lysines and six arginines (amino acids47-57) and a cysteine-rich region which contains seven cysteine residues(amino acids 22-37). The basic region (i.e., amino acids 47-57) isthought to be important for nuclear localization and cell penetration(Ruben et al., J. Virol. 63: 1-8 (1989); Hauber et al., J. Virol. 631181-1187 (1989); Rudolph et al. (2003) 278(13):11411). Thecysteine-rich region mediates the formation of metal-linked dimers invitro (Frankel et al., Science 240: 70-73 (1988); Frankel, et al., Proc.Natl. Acad. Sci USA 85: 6297-6300 (1988)) and is essential for itsactivity as a transactivator (Garcia et al., EMBO J. 7:3143 (1988);Sadaie. et al., J. Virol. 63: 1 (1989)). As in other regulatoryproteins, the N-terminal region can be involved in protection againstintracellular proteases (Bachmair et al., Cell 56: 1019-1032 (1989). Seealso, e.g., Morris, M. C. et al., Nature Biotechnol. 19:1173-1176(2001); Dupont, A. J. and Prochiantz, A., CRC Handbook on CellPenetrating Peptides, Langel, Editor, CRC Press, (2002); Chaloin, L. etal., Biochemistry 36(37):11179-87 (1997); and Lundberg, P. and Langel,U., J. Mol. Recognit. 16(5):227-233 (2003); all publicationsincorporated herein by reference.

In one embodiment of the invention, such a cell penetrating agentcomprises the basic region comprising amino acids 47-57 of the HIV-1 TATpeptide (SEQ ID NO:1). In another embodiment, a cell penetrating agentcomprises the basic region comprising amino acids 48-60 of the HIV-1 TATpeptide (SEQ ID NO:2). In yet another embodiment, a cell penetratingagent comprises the basic region comprising amino acids 49-57, 48-60, or47-57 of the HIV-1 TAT peptide, does not comprise amino acids 22-36 ofthe HIV-1 TAT peptide, and does not comprise amino acids 73-86 of theHIV-1 TAT peptide. In still another embodiment, the specific peptidesset forth in Table 1, below, or fragments thereof, can be used as cellpenetrating agents in the methods and compositions as disclosed herein.

TABLE 1 SEQ ID PEPTIDE SEQUENCE NO: HIV-1 TAT (49- RKKRRQRRR  1 57)HIV-1 TAT (48- GRKKRRQRRRTPQ  2 60) HIV-1 TAT (47- YGRKKRRQRRR  3 57)Kaposi fibroblast AAV ALL PAV LLA LLA  4 growth factor P + VQR KRQ KLMPof caiman MGL GLH LLV LAA ALQ  5 crocodylus Ig(5) GA light chainHIV envelope GAL FLG FLG AAG STM  6 glycoprotein GA + PKS KRK 5(NLS of the gp41 SV40) Drosophila RQI KIW FQN RRM KWK K  7 Antennapediaamide RGD peptide X-RGD-X  8 influenza virus GLFEAIAGFIENGWEGMIDG  9hemagglutinin GGYC envelop glycoprotein transportan AGWT LNS AGY LLG KIN 10 LKA LAA LAK KIL Pre-S-peptide (S)DH QLN PAF 11Somatostatin (tyr- (S)FC YWK TCT 12 3-octreotate) (s) optional Serinefor coupling italic = optional D isomer for stability

In yet another embodiment, an active thiol at the 5′ end of the sensestrand can be coupled to a cysteine reside added to the C terminal endof a cell penetrating agent for delivery into the cytosol (such as afragment of tat or a fragment of the Drosophila Antennapedia peptide).Internalization via these peptides bypasses the endocytic pathway andtherefore removes the danger of rapid degradation in the harsh lysosomalenvironment, and can reduce the concentration required for biologicalefficiency compared to free oligonucleotides.

Other arginine rich peptides are also included for use as cellpenetrating agents as disclosed herein. For example, a TAT analog cancomprise D-amino acids and arginine-substituted TAT (47-60), RNA-bindingpeptides derived from virus proteins such as HIV-1 Rev, and flock housevirus coat proteins, and the DNA binding sequences of leucine zipperproteins, such as cancer-related proteins c-Fos and c-Jun and the yeasttranscription factor GCN4, all of which contain several arginineresidues (see Futaki, et al. (2001) J. Biol Chem 276(8):5836-5840 andFutaki, S. (2002) Intl. Pharm 245(1-2):1-7, which are incorporatedherein by reference). In one embodiment, the arginine rich peptidecontains about 4 to about 11 arginine residues. In another embodiment,the arginine residues are contiguous residues.

In another embodiment, the cell penetrating peptides comprise a membranesignal peptide or membrane translocation sequence capable oftranslocating across the cell membrane. A cell penetrating “signalpeptide” or “signal sequence” refers to a sequence of amino acidsgenerally of a length of about 10 to about 50 or more amino acidresidues, many (typically about 55-60%) residues of which arehydrophobic such that they have a hydrophobic, lipid-soluble portion.Generally, a signal peptide is a peptide capable of penetrating throughthe cell membrane to allow the import and/or export of cellularproteins.

As used herein a “signal sequence”, also known as a “leader sequence”can be used, when desired, to direct the peptide through a membrane of acell. Such a sequence refers to an amino acid sequence which can benaturally present on the peptides of the present invention or providedfrom heterologous sources by recombinant DNA techniques.

Signal peptides can be selected from the SIGPEP database (von Heijne,Protein Sequence Data Analysis 1:4142 (1987); von Heijne and Abrahmsen,L., FEBS Letters 224:439-446 (1989)). Algorithms can also predict signalpeptide sequences for use in the compositions (see, e.g., SIGFIND—SignalPeptide Prediction Server version SignalP V2.0b2, Bendtsen et al.“Improved prediction of signal peptides: SignalP 3.0.”J. Mol. Biol.,340:783-795, 2004; Nielsen et al. “Identification of prokaryotic andeukaryotic signal peptides and prediction of their cleavage sites.”Protein Engineering, 10:1-6, 1997; Bairoch and Boeckmann, “TheSWISS-PROT protein sequence data bank: current status” Nucleic AcidsRes. 22:3578-3580, 1994.). When a specific cell type is to be targeted,a signal peptide used by that cell type can be chosen. For example,signal peptides encoded by a particular oncogene can be selected for usein targeting cells in which the oncogene is expressed. Additionally,signal peptides endogenous to the cell type can be chosen for importingbiologically active molecules into that cell type. Any selected signalpeptide can be routinely tested for the ability to translocate acrossthe cell membrane of any given cell type (see, e.g., U.S. Pat. No.5,807,746, which is incorporated herein in its entirety by reference).Exemplary signal peptide sequences with membrane translocation activityinclude, by way of example and not limitation, those of Karposifibroblast growth factor AAVALLPAVLLALLAPAAADQNQLMP. (SEQ ID NO: 17) ora derivative, variant or fragment thereof.

In another embodiment of the present invention, cell penetrating agentscomprise Herpes Simplex Virus VP22 tegument protein, its analogues,derivatives and variants (Elliott, G. and O'Hare, P., Gene Ther. 6:12-21(1999); Derer, W. et al., J. Mol. Med. 77:609-613 (1999)). VP22, encodedby the UL49 gene, is a structural component of the tegument compartmentof the HSV virus. A composition containing the C-terminal amino acids159-301 of HSV VP22 protein is capable of translocating different typesof cargoes into cells. Translocating activity is observed with a minimalsequence of DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 18).Homologues of VP22 found in herpes viruses are also capable of deliveryof attached compounds of interest across cell membranes (Harms, J. S. etal., J. Virol. 74:3301-3312 (2000); Dorange, F. et al., J. Gen. Virol.81:2219-2230 (2000), which are incorporated herein in their entirety byreference).

In another embodiment the present invention, the cell penetratingpeptides comprise cationic peptides with membrane translocationactivity. Cationic amino acids include for example, but are not limitedto, arginine, lysine, and ornithine. Active peptides with arginine richsequences are present in the Grb2 binding protein, having the sequenceRRWRRWWRRWWRRWRR (SEQ ID NO: 19) (Williams, E. J. et al., J. Biol. Chem.272:22349-22354 (1997)) and polyarginine heptapeptide RRRRRRR (7R) (SEQID NO: 20) (Chen, L. et al., Chem. Biol. 8:1123-1129 (2001); Futaki, S.et al., J. Biol. Chem. 276:5836-5840 (2001); and Rothbard, J. B. et al.,Nat. Med. 6(11):1253-7 (2000) which are incorporated herein in theirentirety by reference). An exemplary cell penetrating peptide of thistype has the sequence RPKKRKVRRR (SEQ ID NO: 21), which is found topenetrate the membranes of a variety of cell types. Also useful arebranched cationic peptides capable of translocation across membranes,including by way of example and not limitation, (KKKK)₂GGC (SEQ IDNO:22), (KWKK)₂GCC (SEQ ID NO: 23), and (RWRR)₂GGC (SEQ ID NO: 24)(Plank, C. et al., Human Gene Ther. 10:319-332 (1999) which areincorporated herein in their entirety by reference).

In a further embodiment, the cell penetrating peptides comprise chimericsequences of cell penetrating peptides that are capable of translocatingacross cell membrane. An exemplary molecule of this type is transportanGALFLGFLGGAAGSTMGAWSQPKSKRKV (SEQ ID NO:25), a chimeric peptide derivedfrom the first twelve amino acids of galanin and a 14 amino acidsequence from mastoporan (Pooga, M et al., Nature Biotechnol. 16:857-861(1998). Analogues of transportans are described in Soomets, U. et al.,Biochim Biophys Acta. 1467(1): 165-76 (2000) and Lindgren, M. et al.Bioconjug Chem. 11 (5):619-26 (2000). An exemplary deletion analogue,transportan-10, has the sequence AGYLLGKINLKALAALAKKIL (SEQ ID NO: 26).

Other types of cell penetrating peptides are the VT5 sequencesDPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID NO: 27), which is an amphipathic,beta-sheet forming peptide (Oehlke, J., FEBS Lett. 415(2):196-9 (1997);unstructured peptides described in Oehlke J., Biochim Biophys Acta.1330(1):50-60 (1997); alpha helical amphipatic peptide with the sequenceKLALKLALKALKAALKLA (SEQ ID NO: 28) (Oehlke, J. et al., Biochim BiophysActa. 1414(1-2):127-39 (1998); sequences based on murine cell adhesionmolecule vascular endothelial cadherin, amino acids 615-632LLIILRRRIRKQAHAHSK (SEQ ID NO: 29) (Elmquist, A. et al., Exp Cell Res.269(2):237-44 (2001); sequences based on third helix of the islet 1 geneenhancer protein RVIRVWFQNKRCKDKK (SEQ ID NO: 30) (Kilk, K. et al.,Bioconjug. Chem. 12(6):911-6 (2001)); amphipathic peptide carrier Pep-1KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31) (Morris, M. C. et al., NatBiotechnol. 19(12):1173-6 (2001)); and the amino terminal sequence ofmouse prion protein MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 32)(Lundberg, P. et al., Biochem. Biophys. Res. Commun. 299(1):85-90(2002)). In some embodiments, the cell penetrating peptides arevariants, fragments of derivatives of SEQ ID NOS: 17 to 32.

In some embodiments of the present invention, a cell penetrating agentdoes not comprise amino acids. In such an embodiment, the cellpenetrating agents is a small molecule or comprises polymers of subunitsother than amino acids. For example such subunits can include, but arenot limited to, hydroxy amino acids, N-methyl-amino acids aminoaldehydes, and the like, which result in polymers with reduced peptidebonds. Other subunit types can be used, depending on the nature of theselected backbone. A variety of backbone types can be used to order andposition the sidechain guanidino and/or amidino moieties, such as alkylbackbone moieties joined by thioethers or sulfonyl groups, hydroxy acidesters (equivalent to replacing amide linkages with ester linkages),replacing the alpha carbon with nitrogen to form an aza analog, alkylbackbone moieties joined by carbamate groups, polyethyleneimines (PEIs),and amino aldehydes, which result in polymers composed of secondaryamines.

A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), ketomethylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO₂RNH), retropeptide (NHCO),transalkene (CR═CH), fluoroalkene (CF═CH), dimethylene (CH₂2CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₂4), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted glycines), and backbones withmalonate and/or gem-diaminoalkyl subunits, for example, as reviewed byFletcher et al. (1998) and detailed by references cited therein. Peptoidbackbones (N-substituted glycines) can also be used. Many of theforegoing substitutions result in approximately isosteric polymerbackbones relative to backbones formed from α-amino acids.

Polymers are constructed by any method known in the art. Exemplarypeptide polymers can be produced synthetically, preferably using apeptide synthesizer (Applied Biosystems Model 433) or can be synthesizedrecombinantly by methods well known in the art.

Alternatively, peptides and polypeptides of the present invention, forexample targeting agents, for example RVG peptide or cell permeablepeptides can be obtained directly by chemical synthesis, e.g., using acommercial peptide synthesizer according to vendor's instructions.Methods and materials for chemical synthesis of polypeptides are wellknown in the art. See, e.g., Merrifield, 1963, “Solid Phase Synthesis,”J. Am. Chem. Soc. 83:2149-2154.

A peptide of the present invention, for example RVG peptide or cellpermeable peptides can be introduced into a cell using conventionaltechniques for transporting proteins into intact cells. In someembodiments, the RVG peptide is conjugated to a cell permeable proteinby fusion as discussed herein, and in some embodiments the conjugate isfurther fused to an internalization peptide sequence, for example aninternalization sequence derived from Antennapedia (Bonfanti et al.,Cancer Res. 57:1442-1446) or to a nuclear localization protein such asHIV TAT peptide (U.S. Pat. No. 5,652,122, which is specificallyincorporated herein in its entirety by reference).

Alternatively, the polypeptides of the present invention, for example anRVG peptide and/or a cell permeable peptide can be expressed in the cellfollowing introduction of a DNA encoding the protein, e.g., a nucleicacid encoding the RVG peptide and/or cell permeable protein. In someembodiments, the nucleic acid comprises the nucleic acid sequenceencoding an RVG peptide and a nucleic acid sequence encoding a cellpermeable protein, for example to generate an RVG peptide-cell permeableprotein fusion protein. In such embodiments, conventional expressionvectors are useful in the methods of as described herein, and in someembodiments the subject can be administered the cells expressing thenucleic acid, or the nucleic acids can be administered directly to thesubject by means commonly known in the art, for example by using acatheter.

In some embodiments, the peptides or peptide constructs as describedherein, for example an RVG peptide and/or a cell permeable peptide orderivatives thereof, are cleavable peptides. A cleavable peptide is apeptide comprising an amino acid sequence that is recognized by aprotease or peptidase or other cleaving agent present in a cell, forexample a target cell, or found in surrounding tissue, or produced by amicrobe capable of establishing an infection in a mammal.

Peptides that are cleavable typically have, but are not required tocomprise one or more amino acids in addition to the amino acidrecognition sequence; for example additional amino acids at the amino-or carboxy terminal, or both, ends of the recognition sequence. Means ofadding amino acids to an amino acid sequence, e.g., in an automatedpeptide synthesizer, as well as means of detecting cleavage of apeptide, e.g., by chromatographic analysis for the amino acid productsof such cleavage, are well known to ordinarily skilled artisans giventhe teachings of this invention. Peptide recognition sequences typicallyrange from about 2 to 20 amino acids in length, and are typicallylocated between the two fragments of the peptide to be cleaved, forexample but not limited to, an RVG peptide and a cell permeable peptide.

The peptides or polypeptides useful in the present invention, forexample RVG peptide and/or a cell permeable peptide or derivativesthereof can be modified at their amino termini, for example, so as toincrease their hydrophilicity. Increased hydrophilicity enhancesexposure of the peptides on the surfaces of lipid-based carriers intowhich the parent peptide-lipid conjugates have been incorporated. Polargroups suitable for attachment to peptides so as to increase theirhydrophilicity are well known, and include, for example and withoutlimitation: acetyl (“Ac”), 3-cyclohexylalanyl (“Cha”), acetyl-serine(“Ac Ser”), acetyl-seryl-serine (“Ac-Ser-Ser-”), succinyl (“Suc”),succinyl-serine (“Suc-Ser”), succinyl-seryl-serine (“Suc-Ser-Ser”),methoxy succinyl (“MeO-Suc”), methoxy succinyl-serine (“MeO-Suc-Ser”),methoxy succinyl-seryl-serine (“MeO-Suc-Ser-Ser”) and seryl-serine(“Ser-Ser-”) groups, polyethylene glycol (“PEG”), polyacrylamide,polyacrylomorpholine, polyvinylpyrrolidine, a polyhydroxyl group andcarboxy sugars, e.g., lactobionic, N-acetyl neuraminic and sialic acids,groups. The carboxy groups of these sugars would be linked to theN-terminus of the peptide via an amide linkage. Presently, the preferredN-terminal modification is a methoxy-succinyl modification.

It will be appreciated that peptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and many amino acids, including the terminalamino acids, can be modified either by natural processes such asglycosylation and other post-translational modifications, or by chemicalmodification techniques which are well known in the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Amongthe known modifications which can be present in polypeptides of thepresent invention are, to name an illustrative few, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of apolynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, 1. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W.H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Sifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

It will also be appreciated, as is well known and as noted above, thatpeptides and polypeptides are not always entirely linear. For instance,polypeptides can be branched as a result of ubiquitination, and they canbe circular, with or without branching, generally as a result ofposttranslational events, including natural processing events and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides can be synthesizedby non translational natural processes and by entirely syntheticmethods.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring and;synthetic polypeptides and such modifications can be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylation host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylation asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification can be presentto the same or varying degree at several sites in a given polypeptide.Also, a given peptide or polypeptide can contain many types ofmodifications.

In some embodiments, N-methyl and hydroxy-amino acids can be substitutedfor conventional amino acids in solid phase peptide synthesis. However,production of polymers with reduced peptide bonds requires synthesis ofthe dimmer of amino acids containing the reduced peptide bond. Suchdimers are incorporated into polymers using standard solid phasesynthesis procedures. Other synthesis procedures are well known in theart.

Effector Agents

The present invention provides a method to deliver effector agentsacross the blood brain barrier using, for example, an RVG peptide orvariant thereof. Effector agents delivered by this approach can include,for example but not limited to, therapeutic agents, diagnostic agentsand imaging agents among others.

In some embodiments, an RVG peptide is conjugated to a carrier particle,and an effector agent is associated with the carrier particle. In someembodiments, the carrier particle is a cell permeable agent (for examplebut not limited to a polymeric arginine residue of varying lengths suchas 9R or 11R as disclosed herein or TAT), and in alternative embodimentsa carrier particle is for example, a liposomal or polymericnanoparticles such as a liposome. In some embodiments, where the carrierparticle is for example a liposome, the carrier particle can furthercomprise cell permeable agents and/or targeting agents.

An “effector agent” as used herein refers to an agent that istransported by the carrier particle and targeting agent (i.e. an RVGpeptide) across the BBB. An effector agent can be a chemical molecule ofsynthetic or biological origin. In some embodiments, an effector agentis generally a molecule that can be used in a pharmaceuticalcomposition, for example the effector agent is a therapeutic agent. Aneffector agent as used herein also refers to any chemical entity orbiological product, or combination of chemical entities or biologicalproducts, administered to a subject to treat or prevent or control adisease or condition, and are herein referred to as “therapeuticagents”.

In alternative embodiments, an effector agents can be a chemical entityor biological product, or combination of chemical entities or biologicalproducts, administered to a subject for imaging purposes in the subject,for example to monitor the presence or progression of disease orcondition, and are herein referred to as “imaging agents” or “diagnosticagents”.

A chemical entity or biological product as disclosed herein ispreferably, but not necessarily a low molecular weight compound, but canalso be a larger compound, or any organic or inorganic molecule,including modified and unmodified nucleic acids such as antisensenucleic acids, RNAi, such as siRNA, shRNA, miRNA, nucleic acidanalogues, miRNA analogues, antigomirs, peptides, peptidomimetics,avimers, receptors, ligands, and antibodies, aptamers, polypeptides oranalogues, derivatives or variants thereof. For example, oligomers ofnucleic acids, amino acids, carbohydrates include without limitationproteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs,lipoproteins, aptamers, and modifications, derivatives and combinationsthereof

A therapeutic agent is an agent useful in the treatment of a disease,disorder or malignancy. In some embodiments, the disease, disorder ormalignancy is a central nervous system (CNS) disorder, for example butnot limited to a neurodegenerative disease. In some embodiment, thedisease or disorder is associated with cells expressing theacetylcholine receptor.

As used herein, the terms “treating” or “treatment” of a disease includepreventing the disease, i.e. preventing clinical symptoms of the diseasein a subject that can be exposed to, or predisposed to, the disease, butdoes not yet experience or display a symptom of the disease; inhibitingthe disease, i.e., arresting the development of the disease or aclinical symptom of the disease; or relieving the disease, i.e., causingregression of the disease or a clinical symptom of the disease.

Effector agents useful in the present invention include, for exampleeffector agents for the treatment of diseases associated with cellsexpressing the a subunit of the acetylcholine receptor, e.g., cellsprotected by the BBB, e.g., neuronal cells, e.g., brain cancer cells.Useful therapeutic agents include nucleic acids such as siRNA, smallmolecule drugs, peptides. More than one therapeutic agent can bedelivered by the delivery agent of the present invention.

Effector agents delivered by the compositions and methods of the presentinvention include siRNAs targeting viruses infecting neuronal cells,such as siRNAs targeting flaviviruses, e.g., Japanese encephalitisvirus, (see U.S. Prov. Appl. 60/723,686 and Kumar et al. PLoS Med. 2006April; 3(4):e96) which are incorporated herein in their entirety byreference, siRNAs targeting herpesviruses, e.g., HSV-1, HSV-2, varicellazoster virus (see U.S. Prov. Appl. 60/687,216 and Palliser et al., 2006,Nature 439, 89-94) which are incorporated herein in their entirety byreference. In one embodiment, the siRNAs targeting herpesvirus targetthe latency associated transcript (e.g., GenBank Accession no. M17921).

Effector agents delivered by the composition and methods as disclosedherein include therapeutic agents for prevention and/or treatment ofbrain tumors. For example, RNAi-induced down-regulation of the oncogenicEGFR can serve as an effective therapy for brain tumors [37]. Asignificant (˜50%) decline in the growth of intracranial gliomas alsohas been attained by RNAi-mediated knockdown of proteases, such as thereceptor-bound urokinase plasminogen activator, cathepsin B, or matrixmetalloprotease-9, which enable tumor progression [36, 87]. Completeregression of the tumor growth was achieved with osmotic minipump-aidedintratumoral infusion of a combination of shRNAs.

Effector agents for the treatment of neuronal diseases can be deliveredby the methods as disclosed herein to treat, for example, neurologicaldisorders and neurodegenerative disease for example but not limited to,Alzheimer's disease, Parkinson's disease, Huntington's disease, A.L.S.,multiple sclerosis, neuro-AIDS, brain cancer, stroke, brain injury,spinal cord injury, autism, lysosomal storage disorders, fragile Xsyndrome, inherited mental retardation, inherited ataxias, blindness,paralysis, stroke, traumatic brain injury and spinal cord injury.

Effector agents delivered by the methods as disclosed herein includesmall molecules chemical and peptides to block intracellular signalingcascades, enzymes (kinases), proteasome function, lipid metabolism, cellcycle and membrane trafficking. Agents delivered by the methods of thepresent invention include agents that promote neuronal growth, e.g.,axonal outgrowth. Such therapeutic agents can be useful in the treatmentof neuronal injury, e.g., spinal cord injury, stroke and traumatic braininjury.

A wide variety of therapeutic agents are available and are encompassedfor use as effector agents, for example but not limited to: proteinneurotrophic factors (for example, nerve growth factor) to treat braininjury, neurological diseases or disorders and neurodegenerativediseases; enzymes to replace enzymatic activities lost through geneticdefects where the loss causes severe metabolic storage diseases such asTay-Sachs disease; neurotransmitters and neuromodulators, such asdopamine and β-endorphin, that would be useful for treating Parkinson'sdisease and intractable pain, respectively, or conditions includingdisorders of movement, cognition, and behavior: antibiotics for treatinginfectious diseases, such as neurosyphilis or AIDS, where penetrationinto the brain of systemically administered antibiotics is presently ablock to treatment; chemotherapeutic agents for treating brain tumorswith agents that do not reach the tumor in sufficient amounts whentolerable doses are administered systemically; and diagnostic agents,such as specific contrast media for brain imaging, that are currentlynot used because of poor penetration into the brain upon systemicadministration.

Further exemplary therapeutic agents useful in the methods as describedherein include for example, but are not limited to; various neurotrophicfactors, growth factors, or neurite inhibitory factors that can helpprevent or repair various forms of neuronal damage caused by CNSdisorders such as neurodegenerative diseases, or by ischemic or hypoxiccrises such as stroke, cardiac arrest, suffocation, blood loss, or othertypes of physical injury or trauma. In some embodiments, therapeuticagents are also various neurotrophic hormones, growth factors, orneurite inhibitory factors that can help stimulate the formation of newsynaptic connections between existing neurons and/or guide the outgrowthof neuronal processes to facilitate some connections and discourageothers. In some subjects, this type of treatment can help facilitate therecovery of nervous function lost due to aging or various diseases. Itcan also help subjects regain muscular, speech, and other functionsafter a stroke, head injury, or other ischemic, hypoxic, excitotoxic, orsimilar crisis.

In further embodiments, therapeutic agents for use as effector agentscan include various types of endocrine, paracrine, and related orsimilar polypeptides that can help treat various glandular,growth-related, maturation-related, sexual, and other disorders.Effector agents can be, for example, polypeptides that increase thequantities of certain neurotransmitter molecules inside the BBB to treatvarious neurodegenerative diseases. For example, polypeptides that canincrease dopamine levels inside the brain (by acting as enzymes,hormones, or release factors, or through various other mechanisms) canbe used to treat Parkinson's disease. Alternately, polypeptides that canincrease acetylcholine levels can be useful for treating Alzheimer'sdisease.

In some embodiments of the present invention, effector agents, forexample therapeutic agents can be any desired entity, e.g. polypeptide,polynucleotide, chemical compound, growth factor, hormone, antibody,cytokine, or the like including entities that cannot pass across theblood-brain barrier by themselves.

Usually, an agent that is a therapeutic agent is useful for treatingneuronal cells or other target cells associated with any neurologicallyrelated disorder. For example, an effector agent to be delivered by thecompositions and methods as disclosed herein can be a pharmaceuticallyactive agent or a combination thereof that at least as part of itsaction targets the central nervous system, olfactory, visual system, orany other system associated with neurologically related disorders.

Examples of such therapeutic agents useful as effector agents are, butare not limited to neurotrophic factor including, without anylimitation, nerve growth factor (NGF), ciliary neurotrophic factor(CNTF), brain-derived neurotrophic factor (DNTF), and glial-derivedneurotrophic factor (GDNF). In another embodiment, effector agent usefulto be delivered by the compositions and methods as disclosed hereininclude, without limitation cardiotrophin-1 (CT1), insulin-like growthfactor-1 (IGF1), transforming growth factor-32 (TGF-32), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF) and interferon α.

In yet another embodiment, the effector agents useful in the methods asdisclosed herein are for example, insulin, glia-derived nexin,gangliosides, phosphatylserine, extracellular matrix remodeling enzymesand their inhibitors, integrins and their ligands, nerve toxins, nervetransmitters, protein chaperones, or protease inhibitors, e.g. serineprotease inhibitors such as 4-(2-aminoethyl)-benzenesulfonyl fluoride(AEBSF) and horseradish-peroxidase (HRP).

In another embodiment, the effector agent, for example a siRNAtherapeutic agent as disclosed herein can be prepared to be delivered ina “prodrug” form. The term “prodrug” indicates a therapeutic agent thatis prepared in an inactive form that is converted to an active form(i.e., drug) within the body or cells thereof by the action ofendogenous enzymes or other chemicals and/or conditions.

According to the present invention, the effector agent of the presentinvention can be transported to various target cells or tissues. Forexample, the effector agent of the present invention can be transportedto any nerve cell, e.g. nerve cell in the central nervous system,olfactory, or visual system. The effector agent of the present inventioncan also be transported to a neurologically related target cell ortissue, e.g. cells or tissues that interact with or are targets of thenervous system.

In some embodiments, the therapeutic agents useful as effector agentsare cytotoxic or growth-suppressing polypeptides that can be used insidethe BBB to treat certain types of cancer or other diseases. Therapeuticagents useful in the present invention include, for example, varioustypes of receptor antagonists, antibodies, and other polypeptides thatcan block or suppress one or more types of neuronal activity and can beused to help control and reduce neuropathic pain, hyperalgesia, andsimilar problems.

In some embodiments, lysosomal storage diseases due to lack of aparticular polypeptide in the CNS can be treated by delivery of an agentcomprising that polypeptide or a mimetic thereof into the CNS by themethods of the present invention.

In further embodiments, infections of the CNS by viruses, prions, orbacteria can be treated by delivering into the CNS effector agents thathelp control or reduce the spread of the infection. For example,delivery of effector agents comprising polypeptides that bind to thereceptors and inhibit virus docking and/or viral transport can be ableto reduce the spread of viruses, such as HIV or HSV within the CNS.Further embodiments include, for example, effector agents which arerecombinant antibodies to antigens within the CNS that can be used tomodulate physiological processes in a beneficial or useful way. Forexample, delivery of recombinant antibodies to myelin associated neuriteinhibitory molecules such as No-Go can be able to enable regrowth andregeneration of CNS nerves, following spinal cord injury and othertraumatic injuries.

In some embodiments, an effector agent is a therapeutic agent for thetreatment of brain tumors and gliomas which can be delivered by themethods as described herein, for example such therapeutic agents arechemotherapy agents. The term “chemotherapeutic agent” or “chemotherapyagent” are used interchangeably herein and refers to an agent that canbe used in the treatment of cancers and neoplasms, for example braincancers and gliomas and that is capable of treating such a disorder. Insome embodiments, a chemotherapeutic agent can be in the form of aprodrug which can be activated to a cytotoxic form. Chemotherapeuticagents are commonly known by persons of ordinary skill in the art andare encompassed for use in the present invention. For example,chemotherapeutic drugs for the treatment of brain tumors and gliomasinclude, but are not limited to: temozolomide (Temodar), procarbazine(Matulane), and lomustine (CCNU). Chemotherapy given intravenously (byIV, via needle inserted into a vein) includes vincristine (Oncovin orVincasar PFS), cisplatin (Platinol), carmustine (BCNU, BiCNU), andcarboplatin (Paraplatin), Mexotrexate (Rheumatrex or Trexall).

The term “effective amount” as used herein refers to the amount oftherapeutic agent of pharmaceutical composition to alleviate at leastsome of the symptoms of the disease or disorder. The term “effectiveamount” includes within its meaning a sufficient amount ofpharmacological composition to provide the desired effect. The exactamount required will vary depending on factors such as the type of tumorto be treated, the severity of the tumor, the drug resistance level ofthe tumor, the species being treated, the age and general condition ofthe subject, the mode of administration and so forth. Thus, it is notpossible to specify the exact “effective amount”. However, for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using only routine experimentation.

In one embodiment, the carrier particle is a liposomal or polymericnanoparticles, for example a liposome. The therapeutic agents can beassociated with nanoparticles such as liposomes by any method known tothe skilled artisan, including encapsulation in the interior,association with the lipid portion of the molecule or association withthe exterior of the liposome. Small molecule drugs soluble in aqueoussolution can be encapsulated in the interior of the liposome. Smallmolecule drugs that are poor soluble in aqueous solution can associatewith the lipid portion of the liposome. siRNAs can associate with theexterior of the liposome. siRNAs can be condensed with cationicpolymers, e.g., PEI, or cationic peptides, e.g., protamines, andencapsulated in the interior of the liposome. Therapeutic peptides canbe encapsulated in the interior of the liposome. In some embodiments,therapeutic peptides, and/or targeting agents, for example, transferrin,insulin like growth factor (IGF) and II and peptidomimetics andantibodies thereof can be covalently attached to the exterior of theliposome.

In one embodiment, an effector agent is a nucleic acid, e.g., plasmid,DNA, shRNA, miRNA, stRNA, siRNA, miRNA mimetic or antigomir. In suchembodiments where the effector agent is a nucleic acid, the carrierparticles associated with the effector agent can be liposomal orpolymeric nanoparticles such as a liposome or in some embodiments thecarrier particle is a peptide, for example, TAT, or an polymericarginine peptide of varying length, such as 11R or 9R as disclosedherein (Melikov et al., Cell Mol Life Sci. 2005; 62: 2739-49).Alternatively, carrier particles useful for transporting nucleic acideffector agents to the BBB include, for example protamines, liposomesand polymers. In another embodiment, the effector agent a siRNA and thecarrier particle is a carrier protein such as an polymeric argininepeptide of varying length, such as 11R or 9R as disclosed herein.

In some embodiments, an effector agent, for example RNA interferingagent and the carrier particle are combined together prior to contactinga biological membrane. Combining the RNA interfering agent and thecarrier particle results in an association of the effector agent and thecarrier particle. In one embodiment, an RNA interfering agent and thecarrier particle are directly linked together, and thus, linkers are notrequired for association of the effector agent, for example RNAinterference molecule) with the carrier particle. In another embodiment,the RNA interfering agent and the carrier polymer are bound together viaelectrostatic bonding.

In some embodiments, the effector agent functions as an RNA interferencemolecule. The term “RNAi” as used herein refers to interfering RNA, orRNA interference molecules are nucleic acid molecules or analoguesthereof for example RNA-based molecules that inhibit gene expression.RNAi refers to a means of selective post-transcriptional gene silencing.RNAi can result in the destruction of specific mRNA, or prevents theprocessing or translation of RNA, such as mRNA.

In some embodiments, an effector agent is a siRNA. The term “shortinterfering RNA” (siRNA), also referred to herein as “small interferingRNA” is defined as an agent which functions to inhibit expression of atarget gene, e.g., by RNAi. An siRNA can be chemically synthesized, itcan be produced by in vitro transcription, or it can be produced withina host cell. siRNA molecules can also be generated by cleavage of doublestranded RNA, where one strand is identical to the message to beinactivated.

In one embodiment, an siRNA effector agent is a double stranded RNA(dsRNA) molecule of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, or 30 nucleotides in length, preferably about 15 to about 28nucleotides, more preferably about 19, 20, 21, 22, 23, 24, or 25nucleotides in length, and more preferably about 19, 20, 21, 22, or 23nucleotides in length, and can contain a 3′ and/or 5′ overhang on eachstrand having a length of about 1, 2, 3, 4, or 5 nucleotides. The lengthof the overhang is independent between the two strands, i.e., the lengthof the over hang on one strand is not dependent on the length of theoverhang on the second strand. Preferably the siRNA is capable ofpromoting RNA interference through degradation or specificpost-transcriptional gene silencing (PTGS) of the target messenger RNA(mRNA).

An siRNAs effector agent for use in the methods as disclosed herein alsoinclude small hairpin (also called stem loop) RNAs (shRNAs). In oneembodiment, these shRNAs are composed of a short, e.g. about 19 to about25 nucleotide, antisense strand, followed by a nucleotide loop of about5 to about 9 nucleotides, and the analogous sense strand. Alternatively,the sense strand can precede the nucleotide loop structure and theantisense strand can follow. These shRNAs can be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501, incorporated by reference herein in itsentirety).

The term “shRNA” as used herein refers to short hairpin RNA whichfunctions as RNAi and/or siRNA species but differs in that shRNA speciesare double stranded hairpin-like structure for increased stability.

In some embodiments, the effector agent is an avimer. Avimers aremulti-domain proteins with binding and inhibiting properties and arecomprised typically of multiple independent binding domains linkedtogether, and as such creates avidity and improved affinity andspecificity as compared to conventional single epitope binding proteinssuch as antibodies. In some embodiments, one can use an avimer that is aprotein or polypeptide that can bind simultaneously to a single proteintarget and/or multiple protein targets, as known as multi-pointattachment in the art. Avimers are useful as therapeutic agents whichfunction son multiple drug targets simultaneously for the progenitorcell and/or treatment of multifactorial diseases or disorders, forexample multifactorial neurodegenerative diseases or neurologicaldisease.

In some embodiments, the effector agent is a antigomir. Antigomirs areoligonucleotides, for example synthetic oligonucleotides capable of genesilencing endogenous miRNAs.

The term “association” or “interaction” as used herein in reference tothe association or interaction of an effector agent, e.g., siRNA, with acarrier particle, refers to any association between the effector agent,e.g., siRNA, with a carrier particle, e.g., a peptide carrier, either bya direct linkage or an indirect linkage. An indirect linkage includes anassociation between a effector agent, e.g., siRNA, and a carrierparticle wherein said effector agent, e.g., siRNA, and said carrierparticle are attached via a linker moiety, e.g., they are not directlylinked. Linker moieties include, but are not limited to, e.g., nucleicacid linker molecules, e.g., biodegradable nucleic acid linkermolecules. A nucleic acid linker molecule can be, for example, a dimer,trimer, tetramer, or longer nucleic acid molecule, for example anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length.

A direct linkage includes any linkage wherein a linker moiety is notrequired. In one embodiment, a direct linkage includes a chemical or aphysical interaction wherein the two moieties, the therapeutic agent,e.g., siRNA, and the carrier particle, interact such that they areattracted to each other. Examples of direct interactions includenon-covalent interactions, hydrophobic/hydrophilic, ionic (e.g.,electrostatic, coulombic attraction, ion-dipole, charge-transfer), Vander Waals, or hydrogen bonding, and chemical bonding, including theformation of a covalent bond. Accordingly, in one embodiment, aneffector agent, e.g., siRNA, and the carrier particle are not linked viaa linker, e.g., they are directly linked. In a further embodiment, thetherapeutic agent, e.g., siRNA, and the carrier particle areelectrostatically associated with each other.

In some embodiments, the effector agent can be an imaging agent. Inorder to function as a suitable effector agent for medical imaging, theeffector agent is useful in a molecular imaging diagnosis procedure, forexample but not limited to, magnetic resonance (MR) imaging. Delivery ofsuch effector agents using the methods and compositions as disclosedherein can enhance the imaging of CNS (i.e. brain and spinal cord)structures and function by MRI or PET for example. Contrast enhancementcan be provided by gadolinium, for example, gadolinium in the form ofGd-DTPA-aminohexanoic acid. Other imaging agents are useful in themethods as disclosed herein include, for example other lanthanide ioncoordination complexes can allow for even greater enhanced relaxation athigher field strength (Aime, S., et al., Chem. Soc. Rev. 27:19-29, 1998;Aime et al., J. Mannet. Reson. Iman. 16:394-406, 2002). Paramagnetic CEST agents are useful as imaging agents in the methods and compositions asdisclosed herein, for example as Eu+3, Tb+3, Dy+3, Er+3, Tm+3, or Yb+3alter tissue contrast via chemical exchange saturation transfer ofpresaturated spins to bulk I water (Elst, L. V., et al., Mann. Reson.Med. 47:1121-1130, 2002). In some embodiments, more than one imagingagent can be used simultaneously in the composition and methods of thepresent invention, with techniques available for attachment of multipleimaging agents, for example Gd-DTPA to proteins to enhance the MR signalknown by persons of ordinary skill in the art. The T1 acceleration andcontrast enhancement of Gd and especially Fe have been shown to saturateat very high field strength, however, while these other lanthanides donot, thus taking full advantage of the increased resolution of very highfield strengths.

In some embodiments, an imaging effector agent is useful as diagnosticagent capable of detection in vivo following administration. Exemplaryimaging effector agents useful for diagnostic purposes include electrondense material, magnetic resonance imaging agents, radiopharmaceuticalsand fluorescent molecules. Radionucleotides useful for imaging includeradioisotopes of copper, gallium, indium, rhenium, and technetium,including isotopes ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ^(99m)Tc, ⁶⁷Ga or ⁶⁸Ga. Imagingagents disclosed by Low et al. in U.S. Pat. No. 5,688,488, incorporatedherein by reference, are also useful in the compositions as disclosedherein.

Accordingly in some embodiments, the methods as disclosed hereinprovides methods that are useful for diagnostic purposes, for examplebut not limited to visualization of plaques and other structures in theCNS of a subject, for example visualization of plaques in the brain ofsubject with Alzheimer's Disease. In further embodiments, thecompositions and methods of the present invention are useful formonitoring the effect of a therapeutic intervention and/or forprognostic purposes. For example, in some embodiments the presentinvention can be used for monitoring the efficacy of a therapeutictreatment in a subject treated with a therapy for Alzheimer's Diseaseand monitoring the reduction of plaques in the subject brain.

Accordingly, as disclosed herein the method provides a means to delivernucleic acids, such as siRNA, nucleic acids, nucleic acid analogues,miRNA, miRNA mimetics, antigomirs and the like to neuronal cells in vivoand in vivo. The methods as disclosed herein are useful for deliveringeffector agents to neuronal cells in vitro, in vivo or ex vivo formultiple purposes, such as (i) research purposes including but notlimited to investigating or studying neuronal function and responses,increasing our understanding of neuronal survival, development, andresponse to agents as well as general neuronal function and neuronaltoxicity assays, and (ii) therapeutic purposes.

Central Nervous System (CNS) Disorders

In some embodiments, the present invention provides methods to delivereffector agents across the blood brain barrier in a subject. In analternative embodiment, the present invention provides methods to treatCNS disorders, the method comprising administering to a subject acomposition comprising an effector agent, a targeting agent (forexample, an RVG peptide or variant or fragment or derivative thereof)and a carrier particle, wherein the effector agent is associated withthe carrier particle. Examples of carrier particles are for example, butnot limited to cell permeable agents and various forms of liposomal orpolymeric nanoparticles such as liposomes. Thus, the present inventionprovides methods to treat CNS disorders, such as neurological disordersand neurodegenerative diseases.

CNS disorders include disorders of the central nervous system as well asdisorders of the peripheral nervous system. CNS disorders include, butare not limited such as neurological disorders, neurodegenerativediseases, brain and spinal cord injuries, cerebrovascular ischemia,neurodegenerative diseases, dementia, traumatic brain injury, stroke,post-stroke, post-traumatic brain injury, small-vessel cerebrovasculardisease, and neurological disorders: for example pugilist, pain,neuropathy, neurotrauma, organophosphate poisoning, depression,schizophrenia, anxiety disorders, epilepsy and bipolar disorder andcognitive-related disorders such as dementia and memory loss.

Examples of neurodegenerative diseases useful to be treated by themethod and compositions as disclosed herein include, for example but notlimited to: amyotrophic lateral sclerosis (ALS), Parkinson's disease,Huntington's disease, Wilson's disease, multi-system atrophy,Alzheimer's disease, Pick's disease, Lewy-body disease,Hallervorden-Spatz disease, torsion dystonia, hereditary sensorimotorneuropathies (HMSN), Gerstmann-Skaussler-Schanker; disease,Creutzfeld-Jakob-disease, Machado-Joseph disease, Friedreich ataxia,Non-Friedreich ataxias, Gilles de la Tourette syndrome, familialtremors, olivopontocerebellar degenerations, paraneoplastic cerebralsyndromes, hereditary spastic paraplegias, hereditary optic neuropathy(Leber), retinitis pigmentosa, Stargardt disease, and Kearns-Sayresyndrome.

Other neurodegenerative disorders include, for example e.g. Alpers'disease, Autosomal Dominant Neurodegenerative Disorder, Batten Disease,Cerebral calcinosis, Cockayne Syndrome, corticobasal ganglionicdegeneration, Dementia with Lewy Bodies, Lewy Body Variant, MultipleSystem Atrophy, Neuronal inkanuclear inclusion disease,Olivopontocerebellar Atrophy, Postpoliomyelitis Syndrome, ProgressiveSupranuclear Palsy, Rett Syndrome, Shy-Drager Syndrome, Tauopathies,Tri-nucleotide repeat diseases, and Tuberous Sclerosis.

Further examples of neurodegenerative disorders, include for examplecerebrovascular accidents (CVA), vascular-related dementia, bovinespongiform encephalopathy (BSE), multiple sclerosis (MS), peripheraldisorders with a CNS component, such as septic shock, hepaticencephalopathy, (diabetic) hypertension, diabetic microangiopathy,sleeping sickness, Whipple disease, Duchenne muscular dystrophy (DMD)and (pre)eclampsia, neuropsychiatric disorders, such as depression,autism, anxiety attention deficit hyperactivity disorder (ADHD), bipolardisorder, schizophrenia and other psychoses.

Other CNS disorders include, for example brain tumors, epilepsy,migraine, narcolepsy, insomnia, chronic fatigue syndrome, mountainsickness, encephalitis, meningitis, AIDS-related dementia.

Parkinson's disease (which is classically characterized chiefly bydepigmentation of the substantia nigra and by the presence of Lewybodies) and Parkinsonian Syndromes (or Parkinsonian disorders) areuseful to be treated by the method and compositions as disclosed herein.Parkinson's disease differs from other parkinsonian disorders based onclinicopathologic criteria. (Dauer and Przedborski (2003), Neuron, 39,889-909). Examples of Parkinsonism syndromes include, for example butnot limited to; Parkinson's disease, Secondary Parkinsonism, a familialneurodegenerative disease or a Parkinsonism plus syndrome.Classification of Parkinsonism is briefly, Primary (idiopathic)Parkinsonism-Parkinson's disease (sporadic, familial), Secondary(acquired, symptomatic) Parkinsonism-infectious (postencephalitic, slowvirus), drug-induced (dopamine antagonists and depletors), Hemiatrophy(hemiparkinsonism), Hydrocephalus (normal pressure hydrocephalus),hypoxia, infectious (postencephalistis), metabolic (parathyroiddysfunction), toxin (MPTP, CO, Mn, Hg. CS2, methanol, ethanol), Trauma(pugilistic encephalopathy), tumor, and vascular (multinfarct state),Heredodegenerative Parkinsonism-Huntington's disease, Wilson's disease,Hallervorden-Spatz disease, Olivopontocerebellar and spinocerebellerdegenerations, neuroacanthocytosis, Lubag (X-linkeddystonia-parkinsonism), and mitochondrial cytopathies with stratialnecrosis Multiple system degenerations (parkinsonismplus)-Cortical-basal ganglionic degeneration, Dementia syndromes(Alzheimer's diseases, diffuse Lewy body disease, frontotemporaldementia), Lytico-Bodig (Guamanian Parkinsonism-dementia-ALS), Multiplesystem atrophy syndromes (striatonigral degeneration, Shy-Dragersyndrome, sporadic olivopontocerebellar degeneration (OPAC), motorneuron disease parkinsonism), Progressive pallidal atrophy, andprogressive supranuclear palsy.

The methods and compositions as disclosed herein are also useful in thetreatment of dementias, for example but not limited to, vasculardementia, dementia with Lewy bodies, frontotemporal dementia andParkinsonism linked to chromosome 17, front and temporal dementias,progressive nuclear palsy, corticobasal degeneration, Huntington'sdisease, thalamic degeneration, Creutzfeldt-Jakob dementia, HIVdementia, schizophrenia with dementia, and Korsakoff's psychosis.

Similarly, cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders(ADD), attention deficit hyperactivity disorders (ADHD), and memorydisturbances in children with reaming disabilities can be treated usingthe methods and compositions as disclosed herein.

The methods and compositions as disclosed herein are also useful intreatment of depression. Depression is characterized by sadness, loss ofinterest in activities, and decreased energy. Other symptoms includeloss of confidence and self-esteem, inappropriate guilt, thoughts ofdeath and suicide, diminished concentration, and disturbance of sleepand appetite. A variety of somatic symptoms can also be present. Thoughdepressive feelings are common, especially after experiencing setbacksin life, depressive disorder is diagnosed only when the symptoms reach athreshold and last at least two weeks. Depression can vary in severityfrom mild to very severe, and includes polar (constant) and unipolar(mainic or bi-polar) depression, as well as seasonal affective disorder(SAD). Depression is typically characterized into eight basic dimensionsi.e., Pessimism, Weak Concentration, Sleep Problems, Anhedonia, Fatigue,Loneliness, Low Self-esteem, and Somatic Complaints to define theprofile of children's and adolescents' depression. Depression can occuras an idiopathic disease (with no somatic disease associated with it),or it can be a psychiatric symptom of a somatic disorder, especially anumber of neurodegenerative disorders.

Depressive disorders (DDs) are frequent psychiatric comorbidities ofneurological disorders like multiple sclerosis, stroke, dementia,migraine, Parkinson's disease, and epilepsy. The clinical manifestationsof DDs in these neurological disorders are identical to those ofidiopathic mood disorders, for example, Multiple Sclerosis, Traumaticbrain injury, stroke: dementia, Alzheimer's disease, Migraine,Parkinson's disease, Epilepsy and Huntington's Disease.

In some embodiments, the methods and compositions as disclosed hereinare also useful for modulating neuronal physiology by delivery of agentsthat alter the expression of neuropeptide genes, for example agents thatact as agonists or antagonists or activate or inhibit genes, for examplegenes that (i) express polypeptides that can block and suppress pain(such as so-called “endorphins”); (ii) genes that express growthfactors; (iii) genes that express polypeptides to promote regenerationor prolong the life-spans of cells; and genes express toxicpolypeptides, such as to kill tumor cells

In some embodiments, the methods and compositions as disclosed hereinare also useful in the treatment of pain and pain disorders. Painincludes, for example, nociceptive pain (pain as a result of injury tobodily tissues), including inflammatory pain, allodynia, hyperallodynia,and neuropathic (pain as a result of abnormalities to nerves, spinalcord and brain), including phantom limb pain, post-therapeuticneuralgia. Pain can be acute or chronic pain, and also includespsychogenic pain (pain related to a physiological disorder). Nociceptivepain includes somatic and visceral pain (for example pancreatits,intestinal cystitis, dysmenorrheal, irritable Bowel syndrome, Crohn'sdisease, biliary colic, urethral colic, myocardial infarction and painsyndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethralsyndrome and protatodynia.). Neuropathic pain includes sympathetic pain.Pain can be associated with CNS disorders, for example, multiplesclerosis, spinal cord injury, sciatica, failed back surgery syndrome,traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, andvascular lesions in the brain and spinal cord (e.g., infarct,hemorrhage, vascular malformation).

Neuropathic pain relates to pathological condition that affects neurons,in a manner that generates unwanted and excessive pain signals. This isoften some anatomical reorganization of the nerve connections within theBBB, such that there is chronic or inappropriate pain response. The term“hyperalgesia” is also used, as a descriptive term that translatesdirectly to “excessive pain” and the term “allodynia” is also used torefer to this condition. Neuropathic pain includes post mastectomy pain,“phantom limb” pain, reflex sympathetic dystrophy (RSD), trigeminalneuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain,cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, diabetic neuropathy, cranial neuralgias,and post-herpetic neuralgia, arachnoiditis, post-infective pain (such asoutbreaks of “shingles”, caused by herpes zoster virus and lingeringchronic pain that arises after a traumatic injury. Pain associated withperipheral nerve damage, central pain (i.e. due to cerebral ischemia)and various chronic pain i.e. lumbago, back pain (low back pain),sciatica inflammatory and/or rheumatic pain. Headache pain (for example,migraine with aura, migraine without aura, and other migrainedisorders), episodic and chronic tension-type headache, tension-typelike headache, cluster headache, and chronic paroxysmal hemicrania.Causalgia is another type of neuropathic pain well as other types ofneuropathic pain are well known by persons of skill in the artNeuropathic pain can range over a very wide span of intensity andstarting at annoying up to excruciating, debilitating and unbearable.

In some embodiments, the methods and compositions as disclosed hereinuseful for treating some cases of learning or memory dysfunction, forexample, such as occur in aging, dementia, after brain trauma or injury,and after various types of major surgery, especially surgery involving acardiopulmonary bypass machine.

In further embodiments, the methods and compositions as disclosed hereinuseful for treating disorders due to excitotoxic damage of neurons, orresulting from diseases or injuries that involve ischemia (inadequateblood flow, as occurs during a stroke or cardiac arrest) or hypoxia(inadequate oxygen supply, as occurs during drowning, carbon monoxidepoisoning, etc.) or traumatic head injury. For example, in animalstudies NGF infusion can slow or reverse the retrograde atrophy ofcholinergic cell bodies and fiber networks and other changes in thecholinergic system that are caused by infarction or measured by infarctvolumes or severity (e.g., Cuello et al 1992). Administration of NGF (orinduction of NGF synthesis in viva by clenbuterol) has been shown toreduce infarct volume in rat models of permanent middle cerebral arteryocclusion (Semkova et al 1999). Other animal data indicates that NGF isable to act after a brain insult to block progression of neuronal damage(e.g., Guegan et al 1998). Accordingly, by delivering agents, forexample NGF to the BBB after a stroke or other brain injury or insult,it likely will be able to reduce the extent and severity of subsequentneuronal loss.

In some embodiments, the methods and compositions as disclosed hereinare also useful to treat pathogen infections of the brain and spinalcord, for example but not limited to pathogen is Meningococci (Neisseriameningitidis) can cause infection of the layers covering the brain andspinal cord (meningitis).

In some embodiments, the methods and compositions as disclosed hereinare also useful in treatment of neurological and psychiatric diseasesassociated with neural cell death include septic shock, intracerebralbleeding, subarachnoidal hemorrhage, multiinfarct dementia, inflammatorydiseases (such as vasculitis, multiple sclerosis, and Guillain-Barre-;syndrome), neurotrauma, peripheral neuropathies, polyneuropathies,epilepsies, schizophrenia, depression, metabolic encephalopathies, andinfections of the central nervous system (viral, bacterial, fungal).

In yet another embodiment, the methods and compositions as disclosedherein are also useful to treat disorders where the tissue affected isin contact with neurons and/or the CNS, for example Anterior HornDiseases including Poliomyelitis, Spinal Muscular Atrophy (e.g.Werding-Hoffman), Muscle Disorders, (e.g. Muscular Dystrophies includingDuchene dystrophy, Becker dystrophy, Limb Girdle dystrophy, CongenitalDystrophy, Facioscapulohumeral dystrophy, Distal dystrophy, andOculopharyngeal dystrophy, Necrotizing Myopathies includingPolymyositis, and Dermatomyositis, Metabolic Myopathies includingMalignant Hyperthermia, Mitochondrial Myopathies, Myotonic Disorders,and Congenital Myopathies), diseases of the Neuromuscular Junction,(e.g. Myasthenia Gravis, and Eaton-Lambert Syndrome), diseases of thePeripheral Nerve, (e.g. Metabolic Neuropathies including DiabetesMellitus, Vitamin deficiency, Uremia, and Porphyria, Toxic Neuropathiesincluding alcohol, vincristine, isoniazid, arsenic, lead, hexane,hexachlorophene, acrylamide, and triethyltin, Vasculitic Neuropathiesincluding Polyarteritis nodosa, Churg-Strauss Syndrome, and Rheumatoidartritis, Inflammatory Neuropathies including Guillain-Barre and ChronicInflammatory demyelinating neuropathy, Hypertrophic Neuropathiesincluding Charcot-Marie-Tooth Disease, Dejerine-Sottas Neuropathy, andRefsum's Disease, Genetic Neuropathies including the various forms ofleukodystrophy, Ataxia-telangiectasia and Giant Axonal Neuropathy,Infectious Neuropathies including Herpes Zoster Neuritis, HerpesSimplex, and Leprosy, Diabetic Neuropathies including Distal symmetricalprimarily sensory neuropathy, Autonomic Neuropathy, Proximalasymmetrical painful primary neuropathy, and Cranial mononeuropathy.

In other embodiments, the methods of the present invention are alsouseful to treat CNS disorders where the CNS disorder is a tumor orcancer. Tumors or cancers of the brain are referred to as a brain tumoror cancer, glioma or oligodenrogliomia and are included as CNS disordersherein.

The term “brain tumor” as used herein is any intracranial tumor createdby abnormal and uncontrolled cell division, normally either found in thebrain itself (neurons, glial cells (astrocytes, oligodendrocytes,ependymal cells), lymphatic tissue, blood vessels), in the cranialnerves (myelin-producing Schwann cells), in the brain envelopes(meninges), skull, pituitary and pineal gland, or spread from cancersprimarily located in other organs (metastatic tumors). Primary (true)brain tumors are commonly located in the posterior cranial fossa inchildren and in the anterior two-thirds of the cerebral hemispheres inadults, although they can affect any part of the brain. Most primarybrain tumors originate from glia (gliomas), astrocytes (astrocytomas),oligodendrocytes (oligodendrogliomas), or ependymal cells (ependymoma).There are also mixed forms, with both an astrocytic and anoligodendroglial cell component. These are called mixed gliomas oroligoastrocytomas. Additionally, mixed glio-neuronal tumors (tumorsdisplaying a neuronal, as well as a glial component, e.g.gangliogliomas, disembryoplastic neuroepithelial tumors) and tumorsoriginating from neuronal cells (e.g. gangliocytoma, centralgangliocytoma) can also be encountered.

Other varieties of primary brain tumors include: primitiveneuroectodermal tumors (PNET, e.g. medulloblastoma, meningiomas,medulloepithelioma, neuroblastoma, retinoblastoma, ependymoblastoma),tumors of the pineal parenchyma (e.g. pineocytoma, pineoblastoma),ependymal cell tumors, choroid plexus tumors, neuroepithelial tumors ofuncertain origin (e.g. gliomatosis cerebri, astroblastoma), etc. Anothertype of primary intracranial tumor is primary cerebral lymphoma, alsoknown as primary CNS lymphoma, which is a type of non-Hodgkin'slymphoma.

As used herein, the term “glioma” refers to a tumor originating in theneuroglia of the brain or spinal cord. Gliomas are derived form theglial cell types such as astrocytes and oligodendrocytes, thus gliomasinclude astrocytomas and oligodendrogliomas, as well as anaplasticgliomas, glioblastomas, and ependymomas. Astrocytomas and ependymomascan occur in all areas of the brain and spinal cord in both children andadults.

In some embodiments, the present invention is useful for treatingneurodegenerative disorders of the CNS (such as Alzheimer's disease), bydelivering agents such as neurotrophic or neuroprotective factors toneurons at risk of degenerating. In some embodiments, delivery ofeffector agents, for example neurotrophic factors to target cells, isfor example but not limited to the affected neurons. As a non-limitingexample, where the treatment is for Alzheimer's Disease, the methods andcompositions can be used to deliver effector agents, for example, to thebasal forebrain cholinergic neurons in a subject having or at risk ofdeveloping Alzheimer's disease. Such a delivery of such therapeuticagents can slow, and potentially halt, the neurodegenerative process. Insome embodiments, the methods and compositions as disclosed herein areuseful for treating trauma or injury to the CNS (such as that whichoccurs in head injury), by delivering an effector agent such as aneuronal growth factor (such as neurotrophic factors) to the targetcells, where the target cells are injured and surviving neurons. As anexample, GDNF can be an effector agent which is delivered to targetcells such as cortical motor neurons for treatment of subjects followingstroke and/or head trauma and/or for the treatment of ALS.

In further embodiments, the present invention can be adapted totreatment of various types of CNS-related neurological disorders ordeficiencies which are correlated with either too little or too much ofsome particular polypeptide. This can be accomplished by using thismethod to deliver an agent into BBB-protected CNS tissue, either: (i) anagent, for example a polypeptide which provides an additional quantityof a polypeptide, to reduce or eliminate a deficiency; or, (ii) anagent, for example an RNAi or polypeptide which blocks, antagonizes, orotherwise suppresses a certain molecule, receptor, or reaction, therebyhelping to controlling a CNS disorder that is caused or characterized bytoo much of a particular molecule.

Therapeutic Administration

In one embodiment, the present invention provides a compositioncomprising a targeting agent, for example an RVG peptide and a carrierparticle, wherein an effector agent is associated with the carrierparticle. In some embodiments, the carrier particle is a liposomal orpolymeric nanoparticles such as a liposome or a cell permeable agent. Insome embodiments, the composition comprises an RVG peptide or fragmentor variant thereof, and a carrier peptide and can further comprise acell permeable agent.

In some embodiments, the composition comprises targeting agents, forexample RVG peptides conjugated to carrier particles, and at least oneeffector agent. In some embodiments, where the composition comprises aplurality of targeting agents and carrier particles, there can bevarious different of targeting agents, which can be conjugated all tothe same type of carrier particle, or different carrier particles. Byway of a non-limiting example, the composition can comprise an RVGpeptide as a targeting agent which is conjugated to a liposomal orpolymeric nanoparticles such as a liposome carrier peptide, and thecomposition can also comprise another targeting agent-carrier particleconjugate comprising a transferrin peptide as the targeting agent and apolymeric arginine residue of various length, such as 9R or 11R asdisclosed herein as the carrier peptide. In other words, the compositioncan comprise a plurality of targeting agent-carrier particle conjugateswith effector agents associated with the carrier particles. Accordingly,in some embodiments the targeting agent and carrier particle of eachtargeting agent-carrier particle conjugate can be the same or differenttypes of targeting agent and carrier particles respectively. In someembodiments, the composition comprises a plurality of targeting agentsconjugated to a plurality of different types of carrier particles. Insome embodiments, any combination of targeting agent can be used withany combination of carrier particle. Accordingly, depending on thecarrier particles present in the composition will also determines thetypes of effector agents also in the composition. As a non-limitingexample, if the composition comprises a targeting agent-carrier particleconjugate comprising a liposome carrier particle, the effector agentassociated with the carrier particle can be, for example a smallmolecule, whereas in alternative embodiments, where the compositioncomprises a targeting agent-carrier particle conjugate comprising apeptide carrier particle, for example 9dR or 11dR, the effector agentassociated with the carrier particle can be, for example a nucleic acidfor example a RNAi.

In further embodiments where the composition comprises a plurality oftargeting agents and carrier particles, the effector agent also presentin the composition that is associated with the carrier particle can alsobe different. For instance, an effector agent associated with thecarrier particle can be a different type of effector agent, for examplenucleic acid effector agent or a peptide effector agent. In someembodiments, the effector agent can be different variant of the sametype of effector agent, for example if the effector agent is a nucleicacid, the composition can comprise both RNA and DNA effector agents. Infurther embodiments, the composition can comprise a plurality ofeffector agents that are variants of the same type of agent, for examplevariants or derivatives of siRNA. By way of a non-limiting example, thecomposition can comprise a plurality of RNAi effector agents thatassociate with the carrier peptide, where the RNAi effector agents aredifferent, for example the RNAi effector agent silences different genetargets or targets different regions on the same gene.

Compositions as disclosed herein wherein the carrier particle is aliposome or polymeric nanoparticle can be administered by any convenientroute, including parenteral, enteral, mucosal, topical, e.g.,subcutaneous, intravenous, topical, intramuscular, intraperitoneal,transdermal, rectal, vaginal, intranasal or intraocular. In oneembodiment, the compositions as disclosed herein are not topicallyadministered. In one embodiment, the delivery is by oral administrationof the composition formulation. In one embodiment, the delivery is byintranasal administration of the composition, especially for use intherapy of the brain and related organs (e.g., meninges and spinalcord). Along these lines, intraocular administration is also possible.In another embodiment, the delivery means is by intravenous (i.v.)administration of the composition, which is especially advantageous whena longer-lasting i.v. formulation is desired. Suitable formulations canbe found in Remington's Pharmaceutical Sciences, 16th and 18th Eds.,Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction toPharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia(1985), each of which is incorporated herein by reference.

The compositions as disclosed herein can be administered inprophylatically or therapeutically effective amounts. The targeteddelivery compositions as disclosed herein can be administered along witha pharmaceutically acceptable carrier. A prophylatically ortherapeutically effective amount means that amount necessary, at leastpartly, to attain the desired effect, or to delay the onset of, inhibitthe progression of, or halt altogether, the onset or progression of theparticular disease or disorder being treated. Such amounts will depend,of course, on the particular condition being treated, the severity ofthe condition and individual patient parameters including age, physicalcondition, size, weight and concurrent treatment. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is preferred generally that amaximum dose be used, that is, the highest safe dose according to soundmedical judgment. It will be understood by those of ordinary skill inthe art, however, that a lower dose or tolerable dose can beadministered for medical reasons, psychological reasons or for virtuallyany other reasons.

The terms “composition” or “pharmaceutical composition” are usedinterchangeably herein and refers to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like. Cellsadministered a composition as disclosed herein can be part of a subject,for example for therapeutic, diagnostic, or prophylactic purposes. Thecells can also be cultured, for example cells as part of an assay forscreening potential pharmaceutical compositions, and the cells can bepart of a transgenic animal for research purposes. In addition,compositions for topical (e.g., oral mucosa, respiratory mucosa) and/ororal administration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

The “pharmaceutically acceptable carrier” means any pharmaceuticallyacceptable means to mix and/or deliver the targeted delivery compositionto a subject. The term “pharmaceutically acceptable carrier” as usedherein means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject agents from one organ, or portion of the body, to another organ,or portion of the body. Each carrier must be “acceptable” in the senseof being compatible with the other ingredients of the formulation and iscompatible with administration to a subject, for example a human. Forthe clinical use of the methods of the present invention, targeteddelivery composition of the invention is formulated into pharmaceuticalcompositions or pharmaceutical formulations for parenteraladministration, e.g., intravenous; mucosal, e.g., intranasal; enteral,e.g., oral; topical, e.g., transdermal; ocular, e.g., via cornealscarification or other mode of administration. The pharmaceuticalcomposition contains a compound of the invention in combination with oneor more pharmaceutically acceptable ingredients. The carrier can be inthe form of a solid, semi-solid or liquid diluent, cream or a capsule.These pharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration.

The term “parenteral administration” and “administered parenterally” asused herein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically, ” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the animal's systemand, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of the pharmaceuticalcomposition comprising the RVG peptide and associated agents of thepresent invention into a subject by a method or route which results inat least partial localization of the agents at a desired site. Theagents of the present invention can be administered by any appropriateroute which results in an effective treatment in the subject.

In the preparation of pharmaceutical formulations containing thetargeted delivery composition of the present invention in the form ofdosage units for oral administration the compound selected can be mixedwith solid, powdered ingredients, such as lactose, saccharose, sorbitol,mannitol, starch, arnylopectin, cellulose derivatives, gelatin, oranother suitable ingredient, as well as with disintegrating agents andlubricating agents such as magnesium stearate, calcium stearate, sodiumstearyl fumarate and polyethylene glycol waxes. The mixture is thenprocessed into granules or pressed into tablets.

Soft gelatin capsules can be prepared with capsules containing a mixtureof the active compound or compounds of the invention in vegetable oil,fat, or other suitable vehicle for soft gelatin capsules. Hard gelatincapsules can contain granules of the active compound. Hard gelatincapsules can also contain the targeted delivery composition includingthe targeting moiety and the carrier particle as well as the therapeuticagent in combination with solid powdered ingredients such as lactose,saccharose, sorbitol, mannitol, potato starch, corn starch,arnylopectin, cellulose derivatives or gelatin.

Dosage units for rectal or vaginal administration can be prepared (i) inthe form of suppositories which contain the active substance mixed witha neutral fat base; (ii) in the form of a gelatin rectal capsule whichcontains the active substance in a mixture with a vegetable oil,paraffin oil or other suitable vehicle for gelatin rectal capsules;(iii) in the form of a ready-made micro enema; or (iv) in the form of adry micro enema formulation to be reconstituted in a suitable solventjust prior to administration.

Liquid preparations for oral administration can be prepared in the formof syrups or suspensions, e.g. solutions or suspensions containing from0.2% to 20% by weight of the active ingredient and the remainderconsisting of sugar or sugar alcohols and a mixture of ethanol, water,glycerol, propylene glycol and polyethylene glycol. If desired, suchliquid preparations can contain coloring agents, flavoring agents,saccharin and carboxymethyl cellulose or other thickening agents. Liquidpreparations for oral administration can also be prepared in the form ofa dry powder to be reconstituted with a suitable solvent prior to use.

Solutions for parenteral administration can be prepared as a solution ofa compound of the invention in a pharmaceutically acceptable solvent,preferably in a concentration from 0.1% to 10% by weight. Thesesolutions can also contain stabilizing ingredients and/or bufferingingredients and are dispensed into unit doses in the form of ampoules orvials. Solutions for parenteral administration can also be prepared as adry preparation to be reconstituted with a suitable solventextemporaneously before use.

The methods of the present invention to deliver the targeted deliverycomposition can also be used to deliver the targeted deliverycomposition orally in granular form including sprayed dried particles,or complexed to form micro or nanoparticles.

Methods for delivery of an agent to a discrete area of the brain arewell known in the art, and can include the use of stereotactic imagingand delivery devices.

The present invention encompasses any suitable method for intracranialadministration of a targeted delivery composition to a selected targettissue, including injection of an aqueous solution of a targeteddelivery composition and implantation of a controlled release system,such as a targeted delivery composition incorporating polymeric implantat the selected target site. Use of a controlled release implant reducesthe need for repeat injections. Intracranial implants are known. Forexample, brachytherapy for malignant gliomas can include stereotaticallyimplanted, temporary, iodine-125 interstitial catheters. Scharfen. C.O., et al., High Activity Iodine-125 Intersitial Implant For Gliomas,Int. J. Radiation Oncology Biol Phys 24(4);583-591:1992. Additionally,permanent, intracranial, low dose 1-125 seeded catheter implants havebeen used to treat brain tumors. Gaspar, et al., Permanent 1-125Implants for Recurrent Malignant Gliomas, Int J Radiation Oncology BiolPhys 43(5);977-982:1999. See also chapter 66, pages 577-580, BellezzaD., et al., Stereotactic Interstitial Brachytherapy, in Gildenberg P. L.et al., Textbook of Stereotactic and Functional Neurosurgery,McGraw-Hill (1998).

Furthermore, local administration of a targeted delivery composition totreat malignant gliomas by interstitial chemotherapy using surgicallyimplanted, biodegradable implants is known. For example, intracranialadministration of 3-bis(chloro-ethyl)-1-nitrosourea (BCNU) (Carmustine)containing polyanhydride waters, has found therapeutic application.Brem, H. et al., J Neuro-Oncology 26:111-123:1995.

A polyanhydride polymer, Gliadel® (Stolle R & D, Inc., Cincinnati, Ohio)a copolymer of poly-carboxyphenoxypropane and sebacic acid in a ratio of20:80 has been used to make implants, intracranially implanted to treatmalignant gliomas. Polymer and BCNU can be co-dissolved in methylenechloride and spray-dried into microspheres. The microspheres can then bepressed into discs 1.4 cm in diameter and 1.0 mm thick by compressionmolding, packaged in aluminum foil pouches under nitrogen atmosphere andsterilized by 2.2 megaRads of gamma irradiation. The polymer permitsrelease of carmustine over a 2-3 week period, although it can take morethan a year for the polymer to be largely degraded. Brem, H., et al,Placebo-Controlled Trial of Safety and Efficacy of IntraoperativeControlled Delivery by Biodegradable Polymers of Chemotherapy forRecurrent Gilomas, Lancet 345; 10081012:1995.

Stereotactic procedures can be used for precise intracranialadministration of targeted delivery composition in aqueous form or as animplant. A cranial neuroblastoma is also treated in this manner. Thus,intracranial administration of a targeted delivery composition can becarried out as follows.

A preliminary MRI scan of the patient can be carried out to obtain thelength of the anterior commissure-posterior commissure line and itsorientation to external bony landmarks. The base of the frame can thenbe aligned to the plane of the anterior commissure-posterior commissureline. CT guidance is used and can be supplemented with ventriculography.The posterior commissure can be visualized on 2-mm CT slices and used asa reference point.

Physiological corroboration of target tissue localization can be by useof high and low frequency stimulation through an electrode accompanyingor incorporated into the long needle syringe used. A thermistorelectrode 1.6 mm in diameter with a 2 mm exposed tip can be used(Radionics, Burlington, Mass.). With electrode high frequencystimulation (75 Hz) paraesthetic responses can be elicited in theforearm and hand at 0.5-1.0 V using a Radionics lesion generator(Radionics Radiofrequency Lesion Generator Model RFG3AV). At lowfrequency (5 Hz) activation or disruption of tremor in the affected limboccurred at 2-3 V. With the methods of the present invention, theelectrode is not used to create a lesion. Following confirmation oftarget tissue localization, the targeted delivery composition can beinjected. A typical injection is the desired number of units (i.e. about0.1 to about 5 units of the targeted delivery composition in about 0.1ml to about 0.5 ml of water or saline. A low injection volume can beused to minimize toxin diffusion away from target. Typically, thetargeted delivery composition effect can be expected to wear off withina few days to about 2-4 months depending on the compound. Thus, analternate targeted delivery composition format, targeted deliverycomposition incorporated within a polymeric implant, can be used toprovide controlled, continuous release of a therapeutic amount of thetargeted delivery composition at the desired location over a prolongedperiod (i.e. from about 1 year to about 6 years), thereby obviating theneed for repeated targeted delivery composition injections.

Several methods can be used for stereotactically guided injection of thetargeted delivery composition to various intracranial targets, such asthe arcuate nucleus (AN) for treatment of acromegaly. Thus astereotactic magnetic resonance (MM) method relying on three-dimensional(3D) T1-weighted images for surgical planning and multiplanarT2-weighted images for direct visualization of the AN, coupled withelectrophysiological recording and injection guidance for AN injectioncan be used. See e.g. Bejjani, B. P., et al., Bilateral SubthalamicStimulation for Parkinson's Disease by Using Three-DimensionalStereotactic Magnetic Resonance Imaging and ElectrophysiologicalGuidance, J Neurosurg 92(4);615-25:2000. The coordinates of the centerof the AN can be determined with reference to the patient's anteriorcommissure-posterior commissure line and a brain atlas.

Electrophysiological monitoring through several parallel tracks can beperformed simultaneously to define the functional target accurately. Thecentral track, which is directed at the predetermined target by using MMimaging, can be selected for neurotoxin injection. No surgicalcomplications are expected.

Computer-aided atlas-based functional neurosurgery methodology can beused to accurately and precisely inject the desired neurotoxin orimplant a neurotoxin controlled release implant. Such methodologiespermit three-dimensional display and real-time manipulation ofhypothalamic structures. Neurosurgical planning with mutuallypreregistered multiple brain atlases in all three orthogonalorientations is therefore possible and permits increased accuracy oftarget definition for targeted delivery composition injection orimplantation, reduced time of the surgical procedure by decreasing thenumber of tracts, and facilitates planning of more sophisticatedtrajectories. See for example Nowinski W. L. et al., Computer-AidedStereotactic Functional Neurosurgery Enhanced by the Use of the MultipleBrain Atlas Database, IEEE Trans Med Imaging 19(1);62-69:2000.

In addition to polymeric implants, osmotic pumps can also be utilizedfor delivery of the targeted delivery composition of the presentinvention by continuous infusion. An osmotic minipump contains ahigh-osmolality chamber that surrounds a flexible, yet impermeable,reservoir filled with the targeted delivery composition-containingvehicle. Subsequent to the subcutaneous implantation of this minipump,extracellular fluid enters through an outer semi-permeable membrane intothe high-osmolality chamber, thereby compressing the reservoir torelease the targeted delivery composition at a controlled,pre-determined rate. The targeted delivery composition, released fromthe pump, is directed via a catheter to a stereotaxically placed cannulafor infusion into the cerebroventricular space, as described herein.

The term “subject” and “individual” are used interchangeably herein, andrefer to an animal, for example a human, to whom treatment, includingprophylactic treatment, with the cells according to the presentinvention, is provided. The term “mammal” is intended to encompass asingular “mammal” and plural “mammals,” and includes, but is notlimited: to humans, primates such as apes, monkeys, orangutans, andchimpanzees; canids such as dogs and wolves; felids such as cats, lions,and tigers; equids such as horses, donkeys, and zebras, food animalssuch as cows, pigs, and sheep; ungulates such as deer and giraffes;rodents such as mice, rats, hamsters and guinea pigs; and bears.Preferably, the mammal is a human subject.

For the methods of the invention, the therapeutically effective amountor dose can be estimated initially from cell culture assays. Then, thedosage can be formulated for use in animal models so as to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic effective amount of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the IC₅₀ and theLD₅₀. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in humans.The dosage can vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval can be adjusted individually to provideplasma levels of the carrier portion containing the targeting and immuneresponse triggering portions. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the carrier portion containing the targeting andimmune response triggering portions can not be related to plasmaconcentration. In such cases, other procedures known in the art can beemployed to determine the correct dosage amount and interval.

The amount of the pharmaceutical composition of the preferredembodiments of the present invention administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

The pharmaceutical composition can, if desired, be presented in asuitable container (e.g., a pack or dispenser device), such as an FDAapproved kit, which can contain one or more unit dosage forms containingthe carrier portion containing the targeting and immune responsetriggering portions.

The method can further comprise administering to a subject a secondtherapy, wherein the second therapy is therapy for the treatment of CNSdisorders, or an anti-cancer therapy, for example an anti-angiogenictherapy, chemotherapy, immunotherapy, surgery, radiotherapy,immunosuppresive agents, or gene therapy with a therapeuticpolynucleotide. The second therapy can be administered to the subjectbefore, during, after or a combination thereof relative to theadministration of the composition of the present invention. Anti-cancertherapies are well known in the art and are encompassed for use in themethods of the present invention. Chemotherapy includes, but is notlimited to an alkylating agent, mitotic inhibitor, antibiotic, orantimetabolite, anti-angliogenic agents eyc. The chemotherapy cancomprise administration of CPT-11, temozolomide, or a platin compound.Radiotherapy can include, for example, x-ray irradiation, w-irradiation,γ-irradiation, or microwaves

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1%.

The invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all referencescited throughout this application, including the U.S. provisionalapplication 60/802,337 as well as the figures and table are incorporatedherein by reference.

EXAMPLES Methods:

Lentiviral experiments. Lentiviruses pseudotyped with VSV-G or RVG weregenerated by cotransfection of the lentiviral vector plasmids along withthe helper plasmid pHR′8.9AVPR (core protein) and either the pVSV-g orpLTR-RVG envelope construct into 293T cells. Culture supernatants wereharvested after 48 h and viral particles concentrated byultracentrifugation. Lentiviruses were spin-infected onto Neuro 2a orHeLa cells in the presence of polybrene and after 48 h, transductionefficiency was determined by analyzing GFP expression by flow cytometry.shFVEJ and shLuc lentiviral constructs and experiments to testprotection against intracranial JEV infections in mice have beenpreviously reported⁸.

Peptides and siRNAs. RVG (YTIWMPENPRPGTPCDIFTNSRGKRASNG) (SEQ ID NO:13); RV-MAT (MNLLRKIVKNRRDEDTQKSSPASAPLDDG) (SEQ ID NO:33); RVG-9dR(YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR) (SEQ ID NO: 34); andRV-MAT-9dR (MNLLRKIVKNRRDEDTQKSSPASAPLDDGGGGRRRRRRRRR) (SEQ ID NO:35)peptides were synthesized and purified by HPLC at the Tufts UniversityCore Facility. RVG and RVMAT peptides were also biotinylated at thecarboxy terminus. siRNAs used in the studies included those targetingGFP (siGFP), firefly luciferase (siLuc), the envelope gene of JEV(siFvEJ) described in Kumar et al⁸, murine Cu—Zn superoxide dismutase(SOD-1)¹⁹, and β-galactosidase (βgal724) bearing a motif that elicitsinterferon production²⁶. For some experiments, siRNA with FITC-label atthe 3′ end of the sense strand was used. siRNAs were obtained fromDharmacon, Inc or synthesized at Samchully Pharm. Co. Ltd, Seoul, Korea.

Peptide binding assay For peptide binding studies, Neuro 2a, HeLa, CHO,293T and BHK21cell lines and single cell suspensions made from freshlyisolated mouse brain, spleen or liver were used. Cells were incubatedwith biotinylated peptides at a 2.5 μM in PBS for 20 min at 4 oC, washed3 times with PBS and then treated with streptavidin-PE (SAPE, BDPharmingen) and analyzed by flow cytometry. For competition experiments,cells were incubated with biotinylated RVG peptide in the absence orpresence of different concentrations of α-bungarotoxin (BTX, Sigma).

SMSA For gel mobility shift assays, 100 pmol siRNA was incubated withpeptides at 1:0.1, 1:1 and 1:10 molar ratios of siRNA:peptide for 15min, electrophoresed on a 2% agarose gel and stained with ethidiumbromide. siRNA without peptide or incubated with RVG-bio (without 9R)served as controls.

Cytotoxicity assay To test the cytotoxicity of RVG-9R/siRNA complexes,Neuro 2a cells (triplicates of 2×10⁵ cells/well in 12-well plates) wereincubated at different concentrations of peptide/siRNA complexes for24-48 h before determining the viability by a standard MTT assay.

siRNA transduction and gene silencing in vitro. Uptake of siRNA intocells was monitored using FITC-labeled siGFP. siRNA (100 pmoles) wasincubated with different concentrations of RVG-9R or RV-MAT-9R in serumfree DMEM for 15min at RT. The complexes were then incubated withNeuro2a and HeLa cells (the cells were plated at 5×10⁴ cells per well in12-well plates the previous day). After 4 h incubation at 37° C. themedium was replaced with 2 ml of fresh medium supplemented with 10%fetal bovine serum (FBS; Hyclone) and the cells were cultured for anadditional 8-10 h before examining by flow cytometry. Transfection withLipofectamine 2000 was done as per the manufacturers' instructions.

To test gene silencing, Neuro 2a cells stably expressing GFP aftertransduction with the pLL3.7 lentiviral vector were incubated with 100pmoles of siGFP complexed with peptides at 10:1 peptide/siRNA ratio andGFP expression analyzed 48 h after transduction.

Animal experiments for testing siRNA delivery and gene silencing.Balb/c, C57BL/6-Tg(ACTB-EGFP)1Osb/J and NOD/SCID were purchased fromJackson Labs and used at 4-6 weeks of age. All mouse experiments hadbeen approved by the CBRI institutional review board and animalinfection experiments were done in a biosafety level 3 animal facilityat the CBRI.

To test peptide uptake by brain cells, 50 μg of biotinylated peptides in0.2 ml PBS were injected into the tail veins of Balb/c mice and 4 hlater, single cell suspension of brains were permeabilized, treated withSAPE and analyzed by flow cytometry. For all siRNA delivery experiments,peptide/siRNA complexes (at a peptide:siRNA molar ratio of 10:1) wereprepared in 100 μl of 5% glucose and injected iv at 50 μgsiRNA/mouse/injection. FITC-siRNA delivery and SOD-1 gene knockdownexperiments were done in Balb/c mice. GFP silencing experiments werecarried out in C57BL/6-Tg(ACTB-EGFP)1Osb/J mice. For testing protectionagainst JEV encephalitis, NOD/SCID mice were intraperitoneallychallenged with 5 LD₅₀ of JEV (LD₅₀ was predetermined using serialdilutions of the virus) 4 h before beginning iv peptide/siRNA treatment.

Staining of brain sections. Mice were injected twice with RVG-9R boundsiRNA-FITC and brains harvested 10-12 hours later. Brains were sectionedfrozen on a sliding microtome to 40 μm thickness and incubated for 48 hat 4₀C with mouse anti-FITC antibodies (Jackson Immuno Research, 20μg/ml) or isotype controls (IgG1 from murine myeloma, Sigma, 20 μg/ml).Sections were washed and FITC immunoreactivity visualized with Alexa 488goat anti-mouse secondary antibodies (1:500, Invitrogen).

Quantitative RT-PCR. Total RNA was isolated form different organs ofpeptide/SOD1 siRNA treated mice using RNeasy RNA isolation kit (Qiagen).The RNA was reverse transcribed using Superscript III and randomhexamers (Invitrogen) according to the manufacturer's protocol.Real-time PCR was performed on 1 μl of complementary DNA, or acomparable amount of RNA without reverse transcriptase, using theQuantiTect™ SYBR Green PCR kit (Qiagen) according to the manufacturer'sinstructions. Amplification conditions were: 40 cycles of denaturationat 95° C. for 20 s, annealing at 60° C. for 30 s, and extension at 69°C. for 30 s using Biorad icycler. Primers for GAPDH and SOD-1 have beenpreviously described (27). Standard curves were generated and therelative amount of mRNA was normalized to GAPDH mRNA. Specificity wasre-verified by melt curve analysis and agarose gel electrophoresis.

Northern blot to detect siRNA. 5 μg of RNA extracted from cellsuspensions by the small RNeasy mini kit (Qiagen,), were electrophoresedon a 15% TBE-UREA PAGE gel (Invitrogen), transferred to a positivelycharged nylon membrane (BrightStar-plus; Ambion) and probed with sensesiRNA probes as described earlier (8).

Western blot analysis. Mouse tissue cell suspensions were homogenized inbuffer containing 25 mM HEPES pH 7.5, 300 mM NaCl, 1.5 mM MgCl2 0.1%Triton X 100, 0.2 mM EDTA and 0.5 mM DTT and protease-inhibitor cocktail(Complete-Mini; Roche Diagnostic). The samples (10 μg protein each) wereelectrophoresed on 15% SDS-polyacrylamide mini gels (Bio-Rad) andtransferred to a polyvinylidene difluoride membrane. The membrane wasprobed with anti-β-actin antibodies (Sigma) or anti-SOD1 antibodies(Stressgen Biotechnologies) and visualized using ECL Western blot system(Pierce Biotechnologies).

IFN response. Balb/c mice were iv injected with 50 μg of either siFvEJor siβgal728 complexed with RVG-9R peptide. siRNA βgal728 complexed toLipofectamine-2000™ served as a positive control. Serum samples obtained7 h after siRNA treatment were tested for interferon-alpha levels usingmouse type-I IFN detection ELISA kit (PBL Biomedical Laboratories),according to the manufacturer's instructions.

Quantification and Statistical analysis. Western blot experiments werequantified by determination of band intensities using ImageJ publicdomain software from the National Institutes of Health(http://rsb.info.nih.gov/ij/). All statistical analysis comparing groupsof mice treated with test and control peptides were performed by one-wayANOVA followed by Bonferroni's post test. P<0.05 was consideredsignificant.

Example 1

Generation of lentiviral vectors for stable expression of shRNAs andtesting their antiviral properties. After testing several viral genetargets, the inventors discovered that a siRNA designed to target theenvelope gene of JEV (FvE^(J), nt 1287-1305 of the genomic RNA) couldafford robust protection against JEV infection in cell lines. For stableendogenous expression of this siRNA, the inventors used the lentiviralvector Lentilox pLL3.7 described by Rubinson et al [88]. This vectorcontains an RNA polymerase III U6 promoter to drive shRNA expression aswell as a GFP reporter gene driven off the CMV promoter to enable easyidentification of transduced cells (FIG. 1). Two complementaryoligonucleotides with the siRNA sequence followed by a 9nt loop, areverse complement of the siRNA and transcription termination signalwere commercially synthesized, annealed and cloned into Xho and Hpadigested pLL3.7 vector. Lentiviral particles were generated in 293 Tcells by transfection with pLL3.7/FvE^(J) or pLL3.7/Luc (encodingluciferase shRNA to serve as control) constructs along with plasmidsencoding VSV-G and delta vpr (to supply envelope and regulatory proteinsin trans) using Fugene reagent. Viral supernatants were harvested 48hours after transfection. The virus was titrated in 293T cells using GFPexpression as a marker and expressed as transduction units (TU)/ml. WhenBHK-21 cells were infected with the lentivirus by spinfection in thepresence of 8 ug/ml polybrene, nearly 100% cells were transduced asevidenced by GFP expression (FIG. 2, left). Stable intracellularproduction of FvE^(J) specific siRNA was ascertained 10 days aftertransduction by Northern analysis using ³²P labeled synthetic siRNAsense strand as probe (FIG. 2, right). To test the ability of shRNA toinhibit JEV replication, stably transduced BHK-21 cells were infectedwith JEV at a multiplicity of infection (moi) of 1 and the viralreplication monitored by flow cytometry after staining with aJEV-specific monoclonal 48 h postinfection. As shown in FIG. 3, comparedwith mock and control luciferase shRNA, FvE^(J) shRNA was able toabrogate JEV infection. That the antiviral effect of FvE mediatedprotection is due to siRNA-induced degradation of viral RNA wasconfirmed in Northern blots using viral cDNA probe (FIG. 4).

In vivo effectiveness of FvE^(J). The inventors also tested the FvE^(J)shRNA for conferring protection against JEV-induced encephalitis. Balb/cmice were injected with 2×10⁵ TU of either luciferase or FvE^(J) shRNAencoding lentiviruses intracranially into the frontal lobe as detailedin Kumar et al [6]. Two days later, the mice were again injected withlentiviruses along with the challenge JE virus at the earlier injectionsite and monitored for survival over time. While all 10 mice injectedwith no or control shRNA died within 7 days, all 10 mice injected withFvE^(J) shRNA survived indefinitely (FIG. 5). The brains of control miceon day 5 after JEV challenge showed the typical histopathologicalfeatures of viral encephalitis with leukocyte infiltration and neuronalapoptosis, while no brain inflammation or neuropathology was observed inthe FvE^(J) shRNA treated mice (FIG. 6). Virus titration of brainhomogenates revealed extremely high levels of viral replication in thecontrol mice, whereas the FvE^(J) shRNA-treated mice remained virus free(FIG. 7).

Synthetic siRNAs can also protect mice from JEV-induced encephalitis.Because synthetic siRNA provides a drug like approach for treatmentwithout the safety concerns associated with integration of lentiviralvectors, the inventors also tested the FvE^(J) siRNA for protection. Toenable uptake of siRNA by brain cells, the inventors complexed thesiFvE^(J) or the control siLuc siRNAs with the cationic lipidformulation, JetSI along with the fusogenic lipiddioleoylphosphatidyl-ethanolamine (DOPE), which has been recently usedto deliver siRNA to brain cells in vivo without toxicity [66]. Afterascertaining that JetSI/DOPE can successfully deliver siFvE^(J) intoNeuro 2a cells to inhibit JEV replication (FIG. 8a ), the inventorsinjected approximately 40 μg (3.2 nmoles) of siRNAs, complexed withJetSI/DOPE intracranially 30 min or 6 h after JEV challenge. While inboth groups, all mice injected with the control siLuc died within 5-7days, all siFvE^(J)-treated mice in both groups survived indefinitely(FIG. 8b ). The siRNA treatment neither induced IFN responsive genes asmeasured by RT-PCR analysis nor led to increased levels of serum IFNlevels as measured by ELISA (data not shown). Moreover, thesiFvE^(J)-treated mice were completely healthy and brain sections taken21 days after challenge showed no histopathological alterations,demonstrating that the treatment was non-toxic (FIG. 6). These resultsshow that a single treatment with siFvE^(J) can protect against fatalencephalitis even when administered after the infection has already beenestablished. A siRNA targeting the envelope gene of WNV (siFvEW) wasalso effective in suppressing WN encephalitis.

A single siRNA targeting a region that is highly conserved amongdifferent flaviviruses can suppress encephalitis induced by both JEV andWNV. In contrast to siFvE^(J) sequence (nt 1287-1305), which is onlyconserved within the strains of JEV, a contiguous sequence in the E genecomprising the cd loop (nt 1307-1328) is essential for mediating viralentry and thus, is highly conserved even at the nucleotide level betweenall sequenced strains of JEV, WNV as well as St. Louis encephalitisvirus [89]. Thus the inventors designed a 21 nt siRNA (siFvE)corresponding to this region. This sequence is identical between the twoviruses except for a single nucleotide mismatch at positions 3 and 21 inthe JEV and WNV target sequence respectively, positions where mutationsare reported to be well tolerated with no significant effect on siRNAefficacy [90]. As shown in FIG. 9a , siFvE^(JW) effectively suppressedboth viruses in the Neuro 2a cell line. Moreover, it was also able toafford 80-100% protection against lethal encephalitis induced by eitherJEV or WNV (FIG. 9b ). These results indicate that by careful design ofconserved target sites, it can be possible to use a single siRNA tosuppress related viruses across species. Importantly, the inventors havediscovered that siRNA treatment after infection can also be effective.The inventors have also observed that siRNA treatment even after 18 h ofinfection protected 80% of mice against encephalitis. However, it wasunable to protect when administered 24 h after infection, when progenyvirus has already infected distant areas of brain.

Pseudotyping the lentivirus with RVG permits neuronal cell-specifictargeting and enhances RNAi effectiveness in the CNS. Pseudotyping thelentivirus with the neurotropic Rabies virus glycoprotein (RVG) insteadof the conventionally used VSV-G allows retrograde axonal transport todistal neurons and results in more extensive spread of the transducedgenes [11]. Moreover, RVG pseudotyping can also allow neuronal cellspecific targeting, which could be an advantage to prevent si/shRNAuptake by irrelevant cells. Thus, the inventors tested lentivirusespseudotyped with either VSV-G or RVG for their ability to deliver shRNAto non-neuronal or neuronal cells. Indeed, whereas the VSV-G pseudotypedlentivirus uniformly transduced both HeLa and the mouse neuroblastomacell line Neuro 2a, RVG pseudotyping allowed transduction exclusively ofNeuro 2a, but not HeLa or BHK-21 non-neuronal cells (FIG. 10). Further,the RVG-pseudotyped shFvE^(J) exhibited a more potent antiviral activitycompared to the corresponding VSV-G pseudotyped lentivirus in that, itabrogated JEV infection in Neuro 2a even at an moi of 50 (highest dosetested) while the protection offered by VSVG-pseudotyped shFvE^(J)diminished at moi's higher than 25. This can be due to differences inthe respective receptor density, enabling better entry of RVGpseudotyped virus in neuronal cells.

The inventors compared the VSV-G- or RVG-pseudotyped shFvE^(J)lentiviruses for relative efficacy in vivo by titrating the dose oflentivirus required to provide complete protection against a lethalintracranial challenge with JEV. Mice received either 2×10⁵ or 2×10³transduction units (TU) of the control shLuc or shFvE^(J), pseudotypedwith either VSV-G or RVG. Mice were challenged with 4 LD₅₀ of JEVinjected at the same site and observed for mortality for 21 days. Allmice injected with the control shLuc, whether pseudotyped with VSV-G orRVG developed encephalitis and succumbed by day 6. In contrast, all micereceiving a high dose of shFvE^(J) lentivirus with either pseudotypingwere protected. However, when a lower dose of lentivirus (2×10³ TU) wasused, all mice receiving VSV-G-pseudotyped shFvE^(J) died, while allmice injected with RVG-pseudotyped shFvE^(J) lentivirus survivedindefinitely (FIG. 11). When the extent of protection was furtheranalyzed by serially increasing the dose of JEV challenge, we could showthat the RVG-pseudotyped lentivirus could protect against JEV challengeat as high a dose as 50 LD₅₀. This enhanced protective efficacy isprobably due to the capacity of RVG-pseudotyped lentivirus to mediateretrograde axonal transport and increase lateral spread from theinjection site [11], resulting in a more extensive protection ofneighboring cells.

A short synthetic peptide derived from RVG specifically binds neuronalcells. The glycoprotein from the neurotropic Rabies virus shows asignificant homology with the snake venom alpha neurotoxin that binds tothe nicotinic acetylcholine receptor [91]. In fact, further studiesshowed that the acetylcholine receptor is also a Rabies virus receptor[92, 93]. Interestingly an RVG peptide was also found to competitivelyinhibit α-bungarotoxin binding to the acetylcholine receptor [94].Structure-function analysis of the binding domain identified a 29 aapeptide (spanning aa 173-202 in the RVG) as sufficient for competitionwith bungarotoxin binding [95]. However, there has been no indicationthat this 29 mer RVG peptide (RVG) (SEQ ID NO:13) binds to neuronalcells that express the a subunit of the acetylcholine receptor, nor thatit can facilitate delivery to such cells. To detect binding, theinventors synthesized a biotinylated 29 mer RVG peptide (SEQ ID NO:13).For control purpose, the inventors also synthesized a 29 mer scrambledRVG peptide (herein termed “RV-MAT”). Acetylcholine expressing Neuro 2a[96, 97] and the receptor negative HeLa cells were incubated with thepeptides, washed and stained with streptavidin PE (SAPE). As shown inFIG. 12, Neuro 2a but not HeLa cells specifically bound the RVG peptidecorresponding to SEQ ID NO:13, but not the scrambled RVG peptide. Tofurther confirm the binding specificity, the inventors tested if abungarotoxin can inhibit RVG peptide binding. Neuro 2a cells were firsttreated with different concentrations (10-9 to 10-3 M) of α-bungarotoxinand then tested for RVG peptide (10-7 M) binding. As shown in FIG. 13,α-bungarotoxin could effectively inhibit RVG peptide binding. Theinventors also tested if the RVG peptide can bind primary neuronal cellsisolated ex vivo from the mouse brain. Single cell suspensions offreshly isolated brain cells and splenocytes (for control) were treatedwith RVG-Bio or a control biotinylated listeria peptide and SAPE andexamined by flow cytometry. Again, the brain cells but not splenocyteswere capable of binding the RVG peptide (FIG. 14). These resultsdemonstrate that the RVG peptide corresponding to SEQ ID NO:13 canspecifically bind primary neuronal cells. Because actelylcholinereceptor α7 subunit is widely expressed by many cell types in the brainincluding the neurons, astrocytes and glia cells as well as the braincapillary endothelial cells [98], the inventors also tested if RVGpeptide delivered intravenously in mice can be taken up by the braincells. Mice were injected IV with 100 μg of biotinylated RVG peptide ora control listeria peptide and 2 h later, single cell suspensions ofbrain examined by flow cytometry after staining with SAPE. As shown inFIG. 15, brain cells from mice treated with RVG but not control peptidewere positive for PE fluorescence, demonstrating that the RVG peptidecan allow transvascular delivery of si/shRNAs.

RVG peptide fused to the cell penetrating HIV TAT peptide can delivershRNA vector to neuronal cells. Although the RVG peptide can bindneuronal cells, it does not bind nucleic acids and therefore cannot beused alone to transport naked nucleic acids, for example si/shRNAs.However, if the RVG peptide corresponding to SEQ ID NO:13 is coated onnanoparticles or liposomes, the RVG peptide can be used to delivernucleic acids to neuronal cells. Alternatively, a peptide from HIV-TATcan bind the negatively charged nucleic acids by charge interaction.HIV-TAT is also useful as it also functions as a cell penetratingpeptide[?-9]' Thus, the inventors synthesized a chimeric RVGTAT peptideconsisting of the 29mer RVG and 15mer TAT peptide. For testing delivery,the inventors first incubated the pLL37 lentiviral vector DNA with theRVG-TAT peptide (5 μg of DNA with 5-10 fold excess of peptide on a molarbasis in a total volume of 0.5 ml DMEM without serum) for 15-20 min andthen, this mixture was added to Neuro 2a or BHK-21 cells. Afterincubating for 4 h, the cells were washed and cultured with serumcontaining culture media. Two days later, the cells were examined forGFP expression by flow cytometry. As shown in FIG. 16, Neuro 2a cellsexpressed GFP at a high level compared to minimal GFP expression byBHK-21 cells. Since BHK-21 cells do not bind the RVG peptide, the smalllevel of GFP expression seen probably represents transduction mediatedby the TAT peptide alone as has been previously reported [7, 8]. Thisdemonstrates that the RVG peptide by enhancing cell binding can greatlyenhance delivery in neuronal cells. Thus, RVG-TAT peptide can also beused for neuronal nucleic acid (such as DNA or RNA) delivery.

RVG peptide fused to the cell penetrating 11dR peptide can deliversynthetic siRNA to neuronal cells. A 9-11 mer of 1-arginine (9R or 11R)peptide has been shown to be 20-fold more efficient than the TAT peptideat cellular uptake, and substitution of 1 with d-arginine was found toenhance uptake by >100 fold [100]. The inventors tested if the RVGpeptide fused to an 11 mer dR (RVG-11dR) could deliver shRNA vector andsiRNAs to neuronal cells. The pLL3.7 shRNA vector or a FITC conjugatedGFP siRNA was bound to RVG-11dR peptide as described for the RVG-TAT andwas used to transduce Neuro 2a cells. GFP expression and siRNA deliverywas determined by flow cytometry 2 days later after thoroughly washingthe cells. As FIG. 17 shows, 11dR was able to deliver both the DNAvector and siRNA efficiently. The inventors also tested if the siRNAdelivered via RVG-11dR can mediate gene silencing. For this, Neuro 2acells were first transduced with a GFP encoding plasmid via RVG-TAT and2 days later, the cells were transduced with anti-GFP siRNA bound toRVG-11dR and GFP expression determined 2 days later. Indeed, RVG-11dRdelivered siRNA could inhibit GFP expression significantly (FIG. 18).

Targeting liposomes. The inventors describe herein the use of RVG-coatedliposomes for in vivo neuronal delivery. A novel nanoscalehyaluronan-coated liposome formulation for nontoxic, targeted in vivodelivery was recently developed, as described in U.S. Provisional Appl.60/723,686, which is incorporated herein in its entirety by reference.Here, the siRNA is encapsulated with a liposome for protection againstenzymes in the body fluids. The liposome is also coated with hyaluran,which enables covalent linkage with targeting molecules such asantibodies or peptides. To specifically target activated T cells, theliposome was coated with the AL-57 antibody that recognizes the form ofLFA-1 that is expressed on activated T cells [101]. CD4 T cells wereactivated with ICAM-1 or αCD3 antibody alone or together. On day 7 afteractivation, the cells were treated with CD4 siRNA (1 nmol) encapsulatedliposomes coated with the LFA-1 or an isotype control antibody and CD4expression determined 2 days later. CD4 expression was specificallyreduced after delivery of CD4 siRNA with LFA-1 antibody coated liposomes(FIG. 19, top panel), but not with control antibody coated liposomes(FIG. 19, middle panel).

Example 2

To address the question of whether different carrier particles could befused to an RVG peptide for delivery of nucleic acids, for example RNAito neuronal cells for functional knock down of gene expression, theinventors used a short peptide derived from the Rabies virus (RVG) thatbinds to the nicotinic acetylcholine receptor as a means of neuronalcell targeting. The RVG was biotinylated to detect neuronalcell-specific binding (as shown in FIG. 20A) and was inhibited byα-bungarotoxin as shown in FIG. 20B. The RVG peptide could also bedetected in the mouse brain 2 hours after intravenous injection (FIG.21). A chimeric peptide, termed “CORVUS” or “RVG-9R” herein comprises anRVG peptide of SEQ ID NO:13 fused to a carrier particle which is a cellpenetrating peptide such as a polymeric arginine residue of variouslengths was used to test siRNA binding and delivery. As used in Example2, CORVUS (an RVG peptide comprising SEQ ID NO:13 fused to a 9 or 11 merof 1-arganine (called R9 or 11R)), was assessed for its ability todeliver shRNA vector and RNAi to neuronal cells in vitro and in vivo andto deliver functional RNAi. CORVUS binds and delivers siRNA to neuronalcells in vitro (FIG. 22), and CORVUS carrying anti-GFP RNAi wasfunctional and silenced GFP expression in GFP expressing Neuro 2a cells(FIG. 23). Furthermore, i.v. injection of CORVUS loaded with FITClabeled RNAi was significantly detected in brain tissue of mice (FIG.24C) compared to the control peptide (FIG. 24A). GFP siRNA complexedwith CORVUS specifically silenced GFP expression in the brain of GFPtransgenic mice after intravenous (i.v.) administration (FIG. 25B), andSOD-1 siRNA complexed with CORVUS specifically silenced mouse SOD1expression at the mRNA level and protein level in the brain (FIG. 29B).

Example 3

Pseudotyping lentivirus with RVG confers neuronal cell-specificity. Theenvelope glycoprotein of Rabies virus (RVG) specifically interacts withthe nicotinic acetylcholine receptor (AchR) on neuronal cells to infectbrain cells^(153, 154). The inventors assessed if pseudotypinglentivirus with RVG, instead of the conventionally used vesicularstomatitis virus glycoprotein (VSV-G) could confer neuronalcell-specificity. GFP encoding lentiviral vector Lentilox pLL3.7¹⁵⁵pseudotyped with either RVG or VSV-G was tested for ability to infectneuronal or non-neuronal cells. While VSV-G pseudotyped lentivirusinfected both cell types, RVG pseudotyping resulted in infectionexclusively of Neuro 2a, but not HeLa cells (FIG. 10). RVG has beenshown to mediate retrograde axonal transport and increase the spread ofa viral vector within the brain¹⁵⁶, the inventors also tested if RVGpseudotyping of pLL3.7 encoding a shRNA (shFvE^(J))¹⁵⁷ targetingJapanese encephalitis virus (JEV) increases its antiviral efficacy.Different concentrations of shFvE^(J) lentivirus, pseudotyped with RVGor VSV-G were tested for protection efficacy in an intracranial (ic) JEVchallenge assay¹⁵⁷. While at a high dose (2×10₅ TU) both lentivirusesequally afforded protection, at a lower dose (2×10₃ TU), all micetreated with RVG pseudotyped lentivirus survived but all those treatedwith VSV-G-pseudotyped lentivirus succumbed to JEV infection (FIG. 11).Collectively, these results suggest that RVG confers neuronalcell-specificity and in addition, by facilitating retro-axonal andtrans-synaptic spread¹⁵⁶ also enhances transduction of neighboringneuronal cells.

Short RVG peptide specifically binds to neuronal cells. To determine ifthe 29 mer RVG peptide specifically binds to neuronal cells that expressAchR, the inventors assessed the ability of a biotinylated 29 mer RVGpeptide and a control peptide of similar length derived from the viralmatrix protein (RV-MAT) to bind to neuronal cells. The snake venom toxinα-bungarotoxin specifically binds AchR, and a short 29 amino acidpeptide derived from RVG (spanning aa 173-202 in the RVG) has beenpreviously reported to competitively inhibit α-bungarotoxin binding tothe AchR in solution¹⁰. The inventors discovered that a 29 mer RVGpeptide specifically binds neuronal cells that express AchR. To detectbinding, the inventors synthesized a biotinylated 29 mer RVG peptide andfor control purpose, a peptide of similar length derived from the viralmatrix protein (RV-MAT). AchR expressing Neuro 2a 11,12 and the receptornegative HeLa cells were incubated with the peptides, washed and stainedwith phycoerythrin-conjugated streptavidin (SAPE). As shown in FIG. 26a, Neuro 2a but not HeLa cells specifically bound the RVG-, but notRV-MAT peptide. RVG peptide also did not bind several other non-neuronalcells including 293T, BHK21 and CHO (FIG. 26b ).

To further confirm AchR-mediated binding specificity, the inventorstested if α-bungarotoxin can inhibit RVG peptide binding to Neuro 2acells. Neuro 2a cells were treated with different concentrations (10⁻¹⁰to 10⁻³M) of α-bungarotoxin along with RVG peptide (2.5 μM). As shown inFIG. 26c , α-bungarotoxin inhibited RVG peptide binding in adose-dependent manner in that, at concentrations of 10⁻⁷M and higher,α-bungarotoxin progressively decreased RVG binding. Moreover,α-bungarotoxin was also able to displace pre-bound RVG from Neuro 2acells (not shown).

The inventors also tested if the RVG peptide can bind primary neuronalcells. Single cell suspensions of freshly isolated mouse brain cellswere treated with RVG-bio or the control RVG-MAT-bio peptides followedby SAPE. Again, brain cells but not splenocytes were capable of bindingthe RVG peptide (FIG. 26d ). Because AchR is also expressed by the braincapillary endothelial cells¹³, the inventors also tested if RVG peptideinjected iv can be taken up by the brain cells. Mice were injected ivwith 50 μg of biotinylated RVG peptide or control RVG-MAT peptide and 4h later, single cell suspensions of brain were examined by flowcytometry after internal staining with SAPE. As shown in FIG. 26e ,brain cells from mice treated with RVG but not the control peptide werepositive for PE fluorescence, demonstrating that the RVG peptide cancross the BBB to enter brain cells.

Chimera RVG-9R peptide can bind and deliver siRNA to neuronal cells.While the RVG peptide can bind neuronal cells, it does not bind nucleicacids and therefore cannot be used alone to transport siRNA. However,short positively charged peptides called cell penetrating peptides (suchas a peptide from HIV-TAT) can bind the negatively charged nucleic acidsby charge interaction and also enable cellular uptake¹⁴⁻¹⁶. A nona(L-arginine) (R9) peptide has been shown to be 20-fold more efficientthan the TAT peptide at cellular uptake, and substitution of 1 withd-arginine was found to enhance uptake by >100 fold¹⁷. Moreover, acholesterol conjugated oligo dR has been used for siRNA delivery tosuppress VEGF gene in a tumor model in mice¹⁸. The inventors tested ifthe RVG peptide fused to 9dR could deliver siRNAs to neuronal cells. Theinventors synthesized an RVG-spacer-9dR (designated RVG-9R) peptide anda control RV-MAT-spacer-9dR (RV-MAT-9R) peptide for these studies. Theability of these peptides to bind siRNA was tested in a gelelectrophoresis mobility shift assay (EMSA). As shown in FIG. 27a , bothRVG-9R and RV-MAT-9R peptides were able to bind siRNA, resulting inquenching of fluorescence and mobility shift in a dose-dependent mannerwith maximal and complete binding at 1:10 molar ratio, demonstratingthat the 9R tagging confers siRNA-binding property to both peptides.Next, the inventors tested if RVG-9R can be used to transduce siRNA intocells. For this, 100 pmoles of FITC conjugated siRNA was complexed withdifferent concentrations of RVG-9R and Neuro 2a cells were incubatedwith the complexes for 4 h, washed and cultured for another 8-10 hoursin fresh media before examining by flow cytometry. As shown in FIG. 27b, RVG-9R was able to transduce siRNA in a dose-dependent manner and apeptide: siRNA molar ratio of 10:1 was optimal for maximal transduction,in agreement with EMSA results shown in FIG. 27a . To determine theneuronal specificity of RVG-9R-mediated siRNA delivery, the inventorstransduced Neuro 2a and HeLa cells using FITC-siRNA complexed withRVG-9R or RV-MAT-9R at the optimal siRNA: peptide ratio, usinglipofectamine as positive control. As shown in FIG. 27c , lipofectamineenabled siRNA uptake by both HeLa and Neuro 2a cells as expected andRV-MAT-9R was unable to transduce either cell type. In contrast, RVG-9Rwas able to transduce Neuro 2a but not HeLa cells to a similar degree aslipofectamine. Thus, RVG-9R allows neuronal cell-specific siRNAdelivery.

Although these results indicate that siRNA can be transduced intoNeuro-2a cells by RVG-9R, siRNA will not be functional and/or effectiveunless it is delivered into the cytoplasm, and therefore it wasimportant to test if the introduced siRNA is functional in silencingspecific gene expression. The inventors then assessed the gene silencingability of the RVG-9R delivered siRNA. Neuro 2a cells stably expressinghigh levels of GFP (by lentiviral introduction of pLL3.7 vector encodingGFP) were transduced with anti-GFP siRNA bound to RVG-9R or RVMAT-9R ortransfected with siRNA using lipofectamine (positive control), and GFPexpression determined 2 days later. RV-MAT-9R complexed with siRNA wasunable to reduce GFP levels, while RVG-9R/siRNA silenced GFP expressionto a similar extent as lipofectamine transfection (FIG. 27d ),demonstrating that the RVG-9R delivered siRNA is indeed functional.RVG-9R/siRNA complex was also found to be non-toxic in a MTT assay (>90%viability 48 h after treatment of Neuro-2a cells with RVG-9R at up to25:1 peptide-siRNA ratio, data not shown).

RVG-9R peptide can deliver siRNA to the brain cells after iv injectionin mice. For potential in vivo delivery, the inventors examined ifRVG-9R binding protects the siRNA against degradation from serumnucleases. Unlike naked siRNA, RVG-9R-bound siRNA was at stable for upto 8 h (FIG. 31).

The inventors assessed if RVG-9R can transport siRNA to brain cells invivo after iv injection. To examine this, the inventors examined if theRVG-9R binding protects siRNA against degradation from serum proteases.Mice were injected iv with 50 μg of FITC-labeled siRNA complexed witheither RVG-9R or the control RV-MAT-9R peptide (at 1:10 molar ratio) ina total volume of 100 μl in 5% glucose solution. The iv injection wasrepeated after 6 hours and 10 hours later, mice were sacrificed andsingle cell suspensions from brain, spleen and liver were examined byflow cytometry for the presence of FITC-positive cells. As shown in FIG.28a , FITC fluorescence was detected in the brain only when the siRNAwas complexed to RVG-9R. However, no FITC uptake was seen in the spleenor liver, demonstrating that RVG-9R allows specific targeting of braincells in vivo. The presence of FITC positive cells in different regionsthroughout the mouse brain was also confirmed by microscopic examinationof brain sections stained with an anti-FITC antibody, as shown in FIG.28 b.

Gene silencing by transvascular delivery of siRNA bound to RVG-9R. Theinventors tested brain-specific gene silencing after iv administrationof RVG-9R/siRNA using GFP Tg mice. GFP siRNA (50 μg) was complexed withRVG-9R or RV-MAT-9R and injected in a volume of 100 μl daily for 3 daysin the tail veins of GFP Tg mice. Two days after the last injection,mice were sacrificed and single cell suspensions of brain, spleen andliver examined for GFP expression by flow cytometry. In GFP Tg mice, theGFP expression was highest in the brain compared to the spleen andliver. Despite this, a significant reduction in GFP expression was seenafter treatment with RVG-9R-bound but not with RV-MAT-bound siRNA (FIG.29b ). Moreover, this gene silencing was only observed in the brain andnot the liver and spleen, demonstrating the specificity of braintargeting. To confirm these results in a different system, the inventorsalso tested silencing of an endogenous gene in the brain followingRVG-9R mediated iv siRNA delivery. In this case, wild type Balb/c micewere iv injected with 50 μg of an siRNA targeting mouse SOD1gene¹⁹complexed to RVG-9R or RV-MAT-9R, 3 times at 8 h intervals and SOD1 mRNAand protein levels in the brain, spleen and liver were measured 24 hafter the last injection by qPCR and Western blot respectively. While nochanges were detected in SOD1 levels in any organ in theRVG-MAT-9R/siRNA treated animals, both SOD1 mRNA and protein levels weresignificantly reduced in the brain, but not other organs in theRVG-9R/siRNA treated mice (FIG. 29b ). To confirm that the observedknockdown is due to the specific delivery of siRNA within the brain, theinventors also tested for the presence of SOD1 siRNA in different organsof mice by Northern blot analysis using siRNA sense strand as probe.siRNA could be detected in the brain but not in the spleen or liver oftreated mice (FIGS. 28a and 29c ). Collectively the inventors havediscovered that RVG-9R is able to deliver siRNA specifically to braincells after iv administration, resulting in effective gene silencing inthe brain.

Example 4

RVG-9R enables i.v treatment of viral encephalitis. The inventors havepreviously reported that intracranial treatment with antiviral siRNAscan provide near complete protection from fatal flaviviral encephalitisin mice⁸. However, a noninvasive iv treatment method would be optimalfor siRNA therapy for clinical use, for example for clinical use inhumans. The inventors demonstrated iv treatment with siRNA bound toRVG-9R can protect mice from fatal JEV-induced encephalitis. Unlike wildtype mice, immunodeficient NOD/SCID mice used in these studies uniformlysusceptible for peripheral infection with flaviviruses^(20, 21).NOD/SCID mice were infected intraperitoneally with 5LD50 (lethal dose)of JEV, followed by after 4 hours an iv injection with 50 μg ofantiviral FvE^(J) siRNA (siFvE^(J) as described in Kumar et al⁸) or ancontrol luciferase siRNA (siLuc) complexed with RVG-9R or RV-MAT-9R. ThesiRNA treatment was repeated every day for at least 3 successive daysand the mice were observed for survival for at least 30 days. Untreatedmice, mice treated with either siFvE^(J) complexed with RV-MAT-9R orwith the control siLuc complexed with RVG-9R all died within 10 days,showing that neither the chimeric peptides by themselves or the controlsiLuc complexed with RVG-9R affected the course of the disease. Incontrast, treatment with siFvE^(J) complexed with RVG-9R resulted in˜80% survival as shown in FIG. 31a , and the surviving mice wereobserved, and did not show any signs of sickness or toxicity during theentire course of observation for 30 days (FIG. 30a ). Moreover, whilethe brain sections of control siRNA treated sick mice revealed typicalfeatures of diffuse focal encephalitis marked by inflammatory cellinfiltration and neuronal apoptosis, similar sections of RVG-9R/siRNAtreated surviving mice were completely normal without anyhistopahological evidence of infection or toxicity (not shown).

The inventors also discovered the presence of siRNA in the brains ofRVG-9R treated mice by Northern blot analysis (FIG. 30b ). The inventorshave also earlier reported that FvE^(J) siRNA effects are indeed due toRNAi and not because of induction of non-specific IFN response⁸.However, to rule out the possibility that non-specific IFN productionmediated the protection observed, and to rule out the possibility thatFvE^(J) siRNA complexed with RVG-9R can induce non-specific IFNresponses, the inventors also measured serum IFN levels followingRVG-9R/siRNA administration. Although, as expected IFN levels wereelevated when mice were treated with a known immunostimulatory siRNA(βgal724)²², IFN was not induced in RVG-9R/FvE^(J) siRNA treated animals(FIG. 30c ), demonstrating the protection is mediated by RNAi. Thus, ivtreatment with RVG-9R/siRNA can be safely and effectively used for thetreatment of fatal JEV infection.

It is likely that the siRNA is widely distributed within the brainbecause most cell types in the brain including neurons, astrocytes andglial cells express the α7 subunit of AchR¹³.

Taken together, the inventors have discovered that an RVG peptideconstruct comprising SEQ ID NO:13 permits transvascular delivery ofsiRNA to the CNS. Although the exact mechanism by which such a construct(for example RVG-9R, RVG-11R or other RVG-containing constructs, orvariants, derivatives or fragments thereof) enable BBB crossing is notknown and not wishing to be bound by theory, because the RVG peptidealone (without 9R) was also able to enter the brain cells after ivinjection (FIG. 27d ), it is likely that receptor-mediated transcytosisvia α7-10 subunit of the AchR (which is widely expressed in the brainincluding expression by brain capillary endothelial cells¹³) is involvedin the process. The fact that RVG-9R, but not RV-MAT-9R was capable offacilitating BBB crossing also indicates that specific receptor bindingcan be important. Although cell penetrating peptides might also enablemembrane crossing of cargo covalently conjugated at the terminus byunknown mechanisms^(23, 24,) receptor clustering mediated by a 10:1molar ratio of siRNA/RVG-9R binding may be important for efficienttransport of siRNA into the neuronal cells (particularly when the siRNAis non-covalently bound to the peptide) and this can explain theneuronal cell specificity of targeting by RVG-9R. Because the RVG-9Rdelivered siRNA was functional in gene silencing in multiple systems, itis likely that the siRNA detaches from the peptide inside the cell.

Similarly, siRNA complexed with protamine has also been reported to bereleased in the cytoplasm of cells by unknown mechanisms to mediate genesilencing²⁵. The inventors have discovered that RVG-9R mediatestransvascular delivery of siRNAs to the CNS. The inventors havediscovered an intravenous RNAi-based treatment approach for flaviviralencephalitis, as well as RVG assisted brain delivery useful for deliveryof a variety of other therapeutic molecules such as gene therapy vectorsand small molecule drugs to the brain for the treatment of a widevariety of neurodegenerative diseases and disorders.

REFERENCES

The references cited below and throughout the specification areincorporated herein by reference in their entirety. All patents andother publications identified are expressly incorporated herein byreference for the purpose of describing and disclosing, for example, themethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

-   1. Lieberman, J., Song, E., Lee, S. K. & Shankar, P. Interfering    with disease: opportunities and roadblocks to harnessing RNA    interference. Trends Mol Med 9, 397-403 (2003).-   2. Berkhout, B. RNA interference as an antiviral approach: targeting    HIV-1. Curr Opin Mol Ther 6, 141-5 (2004).-   1. Joost Haasnoot, P. C., Cupac, D. & Berkhout, B. Inhibition of    virus replication by RNA interference. J Biomed Sci 10, 607-16    (2003).-   2. Gitlin, L. & Andino, R. Nucleic acid-based immune system: the    antiviral potential of mammalian RNA silencing. J Virol 77, 7159-65    (2003).-   3. Wang, Q. C., Nie, Q. H. & Feng, Z. H. RNA interference: antiviral    weapon and beyond. World J Gastroenterol 9, 1657-61 (2003).-   4. Kumar P, Lee S K, Shankar P, Manjunath N. A single siRNA    suppresses encephalitis induced by two flaviviruses. PLoS Medicine.    April; 3(4):e96 (2006).-   5. Gupta, B., Levchenko, T. S. & Torchilin, V. P. Intracellular    delivery of large molecules and small particles by cell-penetrating    proteins and peptides. Adv Drug Deliv Rev 57, 637-51 (2005).-   6. Dietz, G. P. & Bahr, M. Peptide-enhanced cellular internalization    of proteins in neuroscience. Brain Res Bull 68, 103-14 (2005).-   7. Deshayes, S., Morris, M. C., Divita, G. & Heitz, F.    Cell-penetrating peptides: tools for intracellular delivery of    therapeutics. Cell Mol Life Sci 62, 1839-49 (2005).-   8. Melikov, K. & Chernomordik, L. V. Arginine-rich cell penetrating    peptides: from endosomal uptake to nuclear delivery. Cell Mol Life    Sci 62, 2739-49 (2005).-   9. Mazarakis, N. D. et al. Rabies virus glycoprotein pseudotyping of    lentiviral vectors enables retrograde axonal transport and access to    the nervous system after peripheral delivery. Hum Mol Genet 10,    2109-21 (2001).-   10. Tijsterman, M., Ketting, R. F. & Plasterk, R. H. The genetics of    RNA silencing. Annu Rev Genet 36, 489-519 (2002).-   11. Dixon, R. A. Natural products and plant disease resistance.    Nature 411, 843-7 (2001).-   12. Plasterk, R. H. RNA silencing: the genome's immune system.    Science 296, 1263-5 (2002).-   13. Bernstein, E., Denli, A. M. & Hannon, G. J. The rest is silence.    Rna 7, 1509-21 (2001).-   14. Ahlquist, P. RNA-dependent RNA polymerases, viruses, and RNA    silencing. Science 296, 1270-3 (2002).-   15. Mello, C. C. & Conte, D., Jr. Revealing the world of RNA    interference. Nature 431, 338-42 (2004).-   16. Meister, G. & Tuschl, T. Mechanisms of gene silencing by    double-stranded RNA. Nature 431, 343-9 (2004).-   17. Hannon, G. J. & Rossi, J. J. Unlocking the potential of the    human genome with RNA interference. Nature 431, 371-8 (2004).-   18. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate    RNA interference in cultured mammalian cells. Nature 411, 494-8    (2001).-   19. Bertrand, J. R. et al. Comparison of antisense oligonucleotides    and siRNAs in cell culture and in vivo. Biochem Biophys Res Commun    296, 1000-4 (2002).-   20. Gitlin, L., Karelsky, S. & Andino, R. Short interfering RNA    confers intracellular antiviral immunity in human cells. Nature 418,    430-4 (2002).-   21. Bagasra, O. & Prilliman, K. R. RNA interference: the molecular    immune system. J Mol Histol 35, 545-53 (2004).-   22. Barik, S. Control of nonsegmented negative-strand RNA virus    replication by siRNA. Virus Res 102, 27-35 (2004).-   23. Novina, C. D. et al. siRNA-directed inhibition of HIV-1    infection. Nat Med 8, 681-6 (2002).-   24. Song, E. et al. Sustained small interfering RNA-mediated human    immunodeficiency virus type 1 inhibition in primary macrophages. J    Virol 77, 7174-81 (2003).-   25. Lee, S. K. et al. Lentiviral delivery of short hairpin RNAs    protects CD4 T cells from multiple clades and primary isolates of    HIV. Blood (2005).-   26. Jiang, M. & Milner, J. Selective silencing of viral gene    expression in HPV-positive human cervical carcinoma cells treated    with siRNA, a primer of RNA interference. Oncogene 21, 6041-8    (2002).-   27. Bitko, V., Musiyenko, A., Shulyayeva, O. & Barik, S. Inhibition    of respiratory viruses by nasally administered siRNA. Nat Med 11,    50-5 (2005).-   28. Tompkins, S. M., Lo, C. Y., Tumpey, T. M. & Epstein, S. L.    Protection against lethal influenza virus challenge by RNA    interference in vivo. Proc Natl Acad Sci USA 101, 8682-6 (2004).-   29. Palliser, D. et al. An siRNA-based microbicide protects mice    from lethal herpes simplex virus 2 infection. Nature (2005).-   30. Li, B. J. et al. Using siRNA in prophylactic and therapeutic    regimens against SARS coronavirus in Rhesus macaque. Nat Med 11,    944-51 (2005).-   31. Ptasznik, A., Nakata, Y., Kalota, A., Emerson, S. G. &    Gewirtz, A. M. Short interfering RNA (siRNA) targeting the Lyn    kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+)    leukemia cells. Nat Med 10, 1187-9 (2004).-   32. Sumimoto, H. et al. Gene therapy for human small-cell lung    carcinoma by inactivation of Skp-2 with virally mediated RNA    interference. Gene Ther 12, 95-100 (2005).-   33. Duxbury, M. S. et al. Systemic siRNA-mediated gene silencing: a    new approach to targeted therapy of cancer. Ann Surg 240, 667-74;    discussion 675-6 (2004).-   34. Lakka, S. S. et al. Inhibition of cathepsin B and MMP-9 gene    expression in glioblastoma cell line via RNA interference reduces    tumor cell invasion, tumor growth and angiogenesis. Oncogene 23,    4681-9 (2004).-   35. Zhang, Y. et al. Intravenous RNA interference gene therapy    targeting the human epidermal growth factor receptor prolongs    survival in intracranial brain cancer. Clin Cancer Res 10, 3667-77    (2004).-   36. Miller, V. M. et al. Allele-specific silencing of dominant    disease genes. Proc Natl Acad Sci USA 100, 7195-200 (2003).-   37. Miller, V. M., Gouvion, C. M., Davidson, B. L. & Paulson, H. L.    Targeting Alzheimer's disease genes with RNA interference: an    efficient strategy for silencing mutant alleles. Nucleic Acids Res    32, 661-8 (2004).-   38. Xia, H. et al. RNAi suppresses polyglutamine-induced    neurodegeneration in a model of spinocerebellar ataxia. Nat Med 10,    816-20 (2004).-   39. Buckingham, S. D., Esmaeili, B., Wood, M. & Sattelle, D. B. RNA    interference: from model organisms towards therapy for neural and    neuromuscular disorders. Hum Mol Genet 13 Spec No 2, R275-88 (2004).-   40. McCaffrey, A. P. et al. RNA interference in adult mice. Nature    418, 38-9 (2002).-   41. Song, E. et al. RNA interference targeting Fas protects mice    from fulminant hepatitis. Nat Med 9, 347-51 (2003).-   42. Ge, Q. et al. Inhibition of influenza virus production in    virus-infected mice by RNA interference. Proc Natl Acad Sci USA 101,    8676-81 (2004).-   43. Miller, G. Drug targeting. Breaking down barriers. Science 297,    1116-8 (2002).-   44. Schlachetzki, F., Zhang, Y., Boado, R. J. & Pardridge, W. M.    Gene therapy of the brain: the transvascular approach. Neurology 62,    1275-81 (2004).-   45. Doolittle, N. D. et al. Safety and efficacy of a multicenter    study using intraarterial chemotherapy in conjunction with osmotic    opening of the blood-brain barrier for the treatment of patients    with malignant brain tumors. Cancer 88, 637-47 (2000).-   46. Kroll, R. A. & Neuwelt, E. A. Outwitting the blood-brain barrier    for therapeutic purposes: osmotic opening and other means.    Neurosurgery 42, 1083-99; discussion 1099-100 (1998).-   47. Muldoon, L. L. et al. Comparison of intracerebral inoculation    and osmotic blood-brain barrier disruption for delivery of    adenovirus, herpesvirus, and iron oxide particles to normal rat    brain. Am J Pathol 147, 1840-51 (1995).-   48. Borlongan, C. V., Emerich, D. F., Hoffer, B. J. & Bartus, R. T.    Bradykinin receptor agonist facilitates lowdose cyclosporine-A    protection against 6-hydroxydopamine neurotoxicity. Brain Res 956,    211-20 (2002).-   49. Makimura, H., Mizuno, T. M., Mastaitis, J. W., Agami, R. &    Mobbs, C. V. Reducing hypothalamic AGRP by RNA interference    increases metabolic rate and decreases body weight without    influencing food intake. BMC Neurosci 3, 18 (2002).-   50. Xia, H., Mao, Q., Paulson, H. L. & Davidson, B. L.    siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol    20, 1006-10 (2002).-   51. Hommel, J. D., Sears, R. M., Georgescu, D., Simmons, D. L. &    DiLeone, R. J. Local gene knockdown in the brain using    viral-mediated RNA interference. Nat Med 9, 1539-44 (2003).-   52. Van den Haute, C., Eggermont, K., Nuttin, B., Debyser, Z. &    Baekelandt, V. Lentiviral vector-mediated delivery of short hairpin    RNA results in persistent knockdown of gene expression in mouse    brain. Hum Gene Ther 14, 1799-807 (2003).-   53. Naldini, L. et al. In vivo gene delivery and stable transduction    of nondividing cells by a lentiviral vector. Science 272, 263-7    (1996).-   54. Blomer, U. et al. Highly efficient and sustained gene transfer    in adult neurons with a lentivirus vector. J Virol 71, 6641-9    (1997).-   55. Raoul, C. et al. Lentiviral-mediated silencing of SOD1 through    RNA interference retards disease onset and progression in a mouse    model of ALS. Nat Med 11, 423-8 (2005).-   56. Ralph, G. S. et al. Silencing mutant SOD1 using RNAi protects    against neurodegeneration and extends survival in an ALS model. Nat    Med 11, 429-33 (2005).-   57. Cao, L. et al. VEGF links hippocampal activity with    neurogenesis, learning and memory. Nat Genet 36, 827-35 (2004).-   58. Harper, S. Q. et al. RNA interference improves motor and    neuropathological abnormalities in a Huntington's disease mouse    model. Proc Natl Acad Sci USA 102, 5820-5 (2005).-   59. Thakker, D. R., Hoyer, D. & Cryan, J. F. Interfering with the    brain: Use of RNA interference for understanding the pathophysiology    of psychiatric and neurological disorders. Pharmacol Ther (2005).-   60. Marshall, E. Gene therapy. Second child in French trial is found    to have leukemia. Science 299, 320 (2003).-   61. Hacein-Bey-Abina, S. et al. A serious adverse event after    successful gene therapy for X-linked severe combined    immunodeficiency. N Engl J Med 348, 255-6 (2003).-   62. Omi, K., Tokunaga, K. & Hohjoh, H. Long-lasting RNAi activity in    mammalian neurons. FEBS Lett 558, 89-95 (2004).-   63. Isacson, R., Kull, B., Salmi, P. & Wahlestedt, C. Lack of    efficacy of ‘naked’ small interfering RNA applied directly to rat    brain. Acta Physiol Scand 179, 173-7 (2003).-   64. Hassani, Z. et al. Lipid-mediated siRNA delivery down-regulates    exogenous gene expression in the mouse brain at picomolar levels. J    Gene Med 7, 198-207 (2005).-   65. Thakker, D. R. et al. Neurochemical and behavioral consequences    of widespread gene knockdown in the adult mouse brain by using    nonviral RNA interference. Proc Natl Acad Sci USA101, 17270-5    (2004).-   66. Thakker, D. R. et al. siRNA-mediated knockdown of the serotonin    transporter in the adult mouse brain. Mol Psychiatry (2005).-   67. Allerson, C. R. et al. Fully 2′-modified oligonucleotide    duplexes with improved in vitro potency and stability compared to    unmodified small interfering RNA. J Med Chem 48, 901-4 (2005).-   68. Elmen, J. et al. Locked nucleic acid (LNA) mediated improvements    in siRNA stability and functionality. Nucleic Acids Res 33, 439-47    (2005).-   69. Layzer, J. M. et al. In vivo activity of nuclease-resistant    siRNAs. Rna 10, 766-71 (2004).-   70. Li, Z. Y., Mao, H., Kallick, D. A. & Gorenstein, D. G. The    effects of thiophosphate substitutions on native siRNA gene    silencing. Biochem Biophys Res Commun 329, 1026-30 (2005).-   71. Soutschek, J. et al. Therapeutic silencing of an endogenous gene    by systemic administration of modified siRNAs. Nature 432, 173-8    (2004).-   72. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with    ‘antagomirs’. Nature 438, 685-9 (2005).-   73. Dorn, G. et al. siRNA relieves chronic neuropathic pain. Nucleic    Acids Res 32, e49 (2004).-   74. Shi, N. & Pardridge, W. M. Noninvasive gene targeting to the    brain. Proc Natl Acad Sci USA 97, 7567-72 (2000).-   75. Shi, N., Boado, R. J. & Pardridge, W. M. Antisense imaging of    gene expression in the brain in vivo. Proc Natl Acad Sci USA 97,    14709-14 (2000).-   76. Shi, N., Boado, R. J. & Pardridge, W. M. Receptor-mediated gene    targeting to tissues in vivo following intravenous administration of    pegylated immunoliposomes. Pharm Res 18, 1091-5 (2001).-   77. Shi, N., Zhang, Y., Zhu, C., Boado, R. J. & Pardridge, W. M.    Brain-specific expression of an exogenous gene after i.v.    administration. Proc Natl Acad Sci USA 98, 12754-9 (2001).-   78. Zhu, C. et al. Organ-specific expression of the lacZ gene    controlled by the opsin promoter after intravenous gene    administration in adult mice. J Gene Med 6, 906-12 (2004).-   79. Zhang, Y., Schlachetzki, F. & Pardridge, W. M. Global non-viral    gene transfer to the primate brain following intravenous    administration. Mol Ther 7, 11-8 (2003).-   80. Zhang, Y., Boado, R. J. & Pardridge, W. M. In vivo knockdown of    gene expression in brain cancer with intravenous RNAi in adult rats.    J Gene Med 5, 1039-45 (2003).-   81. B. N. Fields, D. M. K., P. M. Howley (ed.) Field's Virology,    931-952 (Lippincott-Raven, Philadelphia, 1996).-   82. Biggerstaff, B. J. & Petersen, L. R. Estimated risk of    transmission of the West Nile virus through blood transfusion in the    US, 2002. Transfusion 43, 1007-17 (2003).-   83. Tyler, K. L. West Nile virus infection in the United States.    Arch Neurol 61, 1190-5 (2004).-   84. Solomon, T. Flavivirus encephalitis. N Engl J Med 351, 370-8    (2004).-   85. Gondi, C. S. et al. RNAi-mediated inhibition of cathepsin B and    uPAR leads to decreased cell invasion, angiogenesis and tumor growth    in gliomas. Oncogene 23, 8486-96 (2004).-   86. Rubinson, D. A. et al. A lentivirus-based system to functionally    silence genes in primary mammalian cells, stem cells and transgenic    mice by RNA interference. Nat Genet 33, 401-6 (2003).-   87. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C.    The envelope glycoprotein from tickborne encephalitis virus at 2 A    resolution. Nature 375, 291-8 (1995).-   88. Du, Q., Thonberg, H., Wang, J., Wahlestedt, C. & Liang, Z. A    systematic analysis of the silencing effects of an active siRNA at    all single-nucleotide mismatched target sites. Nucleic Acids Res 33,    1671-7 (2005).-   89. Lentz, T. L., Wilson, P. T., Hawrot, E. & Speicher, D. W. Amino    acid sequence similarity between rabies virus glycoprotein and snake    venom curaremimetic neurotoxins. Science 226, 847-8 (1984).-   90. Lentz, T. L., Burrage, T. G., Smith, A. L., Crick, J. &    Tignor, G. H. Is the acetylcholine receptor a rabies virus receptor?    Science 215, 182-4 (1982).-   91. Lafon, M. Rabies virus receptors. J Neurovirol 11, 82-7 (2005).-   92. Lentz, T. L. Rabies virus binding to an acetylcholine receptor    alpha-subunit peptide. J Mol Recognit 3, 82-8 (1990).-   93. Lentz, T. L. Structure-function relationships of curaremimetic    neurotoxin loop 2 and of a structurally similar segment of rabies    virus glycoprotein in their interaction with the nicotinic    acetylcholine receptor. Biochemistry 30, 10949-57 (1991).-   94. Notter, M. F. & Leary, J. F. Flow cytometric analysis of tetanus    toxin binding to neuroblastoma cells. J Cell Physiol 125, 476-84    (1985).-   95. Chen, T. J., Chen, S. S., Wu, R. E., Wang, D. C. & Lin, C. H.    Implication of nNOS in the enlargement of AChR aggregates but not    the initial aggregate formation in a novel coculture model. Chin J    Physiol 48, 129-38 (2005).-   96. Gotti, C. & Clementi, F. Neuronal nicotinic receptors: from    structure to pathology. Prog Neurobiol 74, 363-96 (2004).-   97. Chiu, Y. L., Ali, A., Chu, C. Y., Cao, H. & Rana, T. M.    Visualizing a correlation between siRNA localization, cellular    uptake, and RNAi in living cells. Chem Biol 11, 1165-75 (2004).-   98. Wender, P. A. et al. The design, synthesis, and evaluation of    molecules that enable or enhance cellular uptake: peptoid molecular    transporters. Proc Natl Acad Sci USA 97, 13003-8 (2000).-   99. Lu, C., Shimaoka, M., Salas, A. & Springer, T. A. The binding    sites for competitive antagonistic, allosteric antagonistic, and    agonistic antibodies to the domain of integrin LFA-1. J Immunol 173,    3972-8 (2004).-   100. Donnelly-Roberts, D. L. & Lentz, T. L. Structural and    conformational similarity between synthetic peptides of    curaremimetic neurotoxins and rabies virus glycoprotein. Brain Res    Mol Brain Res 11, 107-13 (1991).-   101. Dietzschold, B., Schnell, M. & Koprowski, H. Pathogenesis of    rabies. Curr Top Microbiol Immunol 292, 45-56 (2005).-   102. Plakhov, I. V., Arlund, E. E., Aoki, C. & Reiss, C. S. The    earliest events in vesicular stomatitis virus infection of the    murine olfactory neuroepithelium and entry of the central nervous    system. Virology 209, 257-62 (1995).-   103. Lafay, F. et al. Spread of the CVS strain of rabies virus and    of the avirulent mutant AvO1 along the olfactory pathways of the    mouse after intranasal inoculation. Virology 183, 320-30 (1991).-   104. Kilic, E., Kilic, U. & Hermann, D. M. TAT fusion proteins    against ischemic stroke: Current status and future perspectives.    Front Biosci 11, 1716-21 (2006).-   105. Diem, R. et al. HIV-Tat-mediated Bcl-XL delivery protects    retinal ganglion cells during experimental autoimmune optic    neuritis. Neurobiol Dis 20, 218-26 (2005).-   106. Yin, W. et al. TAT-mediated delivery of Bcl-xL protein is    neuroprotective against neonatal hypoxicischemic brain injury via    inhibition of caspases and AIF. Neurobiol Dis (2005).-   107. Pesola, J. M., Zhu, J., Knipe, D. M. & Coen, D. M. Herpes    simplex virus 1 immediate-early and early gene expression during    reactivation from latency under conditions that prevent infectious    virus production. J Virol 79, 14516-25 (2005).-   108. Shah, K. et al. Glioma therapy and real-time imaging of neural    precursor cell migration and tumor regression. Ann Neurol 57, 34-41    (2005).-   109. Shah, K. et al. In vivo imaging of HIV protease activity in    amplicon vector-transduced gliomas. Cancer Res 64, 273-8 (2004).-   110. Tang, Y. et al. In vivo tracking of neural progenitor cell    migration to glioblastomas. Hum Gene Ther 14, 1247-54 (2003).-   111. Shah, K., Tang, Y., Breakefield, X. & Weissleder, R. Real-time    imaging of TRAIL-induced apoptosis of glioma tumors in vivo.    Oncogene 22, 6865-72 (2003).-   112. Schiffelers, R. M. et al. Cancer siRNA therapy by tumor    selective delivery with ligand-targeted sterically stabilized    nanoparticle. Nucleic Acids Res 32, e149 (2004).-   113. Zhang, W. et al. Inhibition of respiratory syncytial virus    infection with intranasal siRNA nanoparticles targeting the viral    NS1 gene. Nat Med 11, 56-62 (2005).-   114. Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., Davis, M. E.    & Triche, T. J. Sequence-specific knockdown of EWS-FLI1 by targeted,    nonviral delivery of small interfering RNA inhibits tumor growth in    a murine model of metastatic Ewing's sarcoma. Cancer Res 65, 8984-92    (2005).-   115. Kulkarni, R. P., Mishra, S., Fraser, S. E. & Davis, M. E.    Single cell kinetics of intracellular, nonviral, nucleic acid    delivery vehicle acidification and trafficking. Bioconjug Chem 16,    986-94 (2005).-   116. Davis, M. E. & Brewster, M. E. Cyclodextrin-based    pharmaceutics: past, present and future. Nat Rev Drug Discov 3,    1023-35 (2004).-   117. Bellocq, N. C. et al. Synthetic biocompatible    cyclodextrin-based constructs for local gene delivery to improve    cutaneous wound healing. Bioconjug Chem 15, 1201-11 (2004).-   118. Pun, S. H. et al. Cyclodextrin-modified polyethylenimine    polymers for gene delivery. Bioconjug Chem 15, 831-40 (2004).-   119. Pun, S. H. et al. Targeted delivery of RNA-cleaving DNA enzyme    (DNAzyme) to tumor tissue by transferrin-modified,    cyclodextrin-based particles. Cancer Biol Ther 3, 641-50 (2004).-   120. Davis, M. E. et al. Self-assembling nucleic acid delivery    vehicles via linear, water-soluble, cyclodextrincontaining polymers.    Curr Med Chem 11, 179-97 (2004).-   121. Bellocq, N. C., Pun, S. H., Jensen, G. S. & Davis, M. E.    Transferrin-containing, cyclodextrin polymerbased particles for    tumor-targeted gene delivery. Bioconjug Chem 14, 1122-32 (2003).-   122. Metselaar, J. M. & Storm, G. Liposomes in the treatment of    inflammatory disorders. Expert Opin Drug Deliv 2, 465-76 (2005).-   123. Torchilin, V. P. Recent advances with liposomes as    pharmaceutical carriers. Nat Rev Drug Discov 4, 145-60 (2005).-   124. Peer, D., Florentin, A. & Margalit, R. Hyaluronan is a key    component in cryoprotection and formulation of targeted unilamellar    liposomes. Biochim Biophys Acta 1612, 76-82 (2003).-   125. Peer, D. & Margalit, R. Physicochemical evaluation of a    stability-driven approach to drug entrapment in regular and in    surface-modified liposomes. Arch Biochem Biophys 383, 185-90 (2000).-   126. Peer, D. & Margalit, R. Loading mitomycin C inside long    circulating hyaluronan targeted nanoliposomes increases its    antitumor activity in three mice tumor models. Int J Cancer 108,    780-9 (2004).-   127. Peer, D. & Margalit, R. Tumor-targeted hyaluronan nanoliposomes    increase the antitumor activity of liposomal Doxorubicin in    syngeneic and human xenograft mouse tumor models. Neoplasia 6,    343-53 (2004).-   128. Zhang, Y., Schlachetzki, F., Li, J. Y., Boado, R. J. &    Pardridge, W. M. Organ-specific gene expression in the rhesus monkey    eye following intravenous non-viral gene transfer. Mol Vis 9, 465-72    (2003).-   129. Huber, J. D., Egleton, R. D. & Davis, T. P. Molecular    physiology and pathophysiology of tight junctions in the blood-brain    barrier. Trends Neurosci 24, 719-25 (2001).-   130. Akdemir, H., Selcuklu, A., Pasaoglu, A., Oktem, I. S. &    Kavuncu, I. Treatment of severe intraventricular hemorrhage by    intraventricular infusion of urokinase. Neurosurg Rev 18, 95-100    (1995).-   131. Vincken, W., Meysman, M., Verbeelen, D., Lauwers, S. &    D'Haens, J. Intraventricular rifampicin in severe tuberculous    meningo-encephalitis. Eur Respir J 5, 891-3 (1992).-   132. Elgamal, E. A. & Murshid, W. R. Intracavitary administration of    amphotericin B in the treatment of cerebral aspergillosis in a non    immune-compromised patient: case report and review of the    literature. Br J Neurosurg 14, 137-41 (2000).-   133. Rawicki, B. Treatment of cerebral origin spasticity with    continuous intrathecal baclofen delivered via an implantable pump:    long-term follow-up review of 18 patients. J Neurosurg 91, 733-6    (1999).-   134. Kamensek, J. Continuous intrathecal baclofen infusions. An    introduction and overview. Axone 20, 93-8 (1999).-   135. Dario, A. & Tomei, G. A benefit-risk assessment of baclofen in    severe spinal spasticity. Drug Saf 27, 799-818 (2004).-   136. Uhrbom, L., Nerio, E. & Holland, E. C. Dissecting tumor    maintenance requirements using bioluminescence imaging of cell    proliferation in a mouse glioma model. Nat Med 10, 1257-60 (2004).-   137. Saydam, O. et al. Herpes simplex virus 1 amplicon    vector-mediated siRNA targeting epidermal growth factor receptor    inhibits growth of human glioma cells in vivo. Mol Ther 12, 803-12    (2005).-   138. Bloom, D. C. HSV LAT and neuronal survival. Int Rev Immunol 23,    187-98 (2004).-   139. Yarom, N., Buchner, A. & Dayan, D. Herpes simplex virus    infection: part I—Biology, clinical presentation and latency. Refuat    Hapeh Vehashinayim 22, 7-15, 84 (2005).-   140. Bystricka, M. & Russ, G. Immunity in latent Herpes simplex    virus infection. Acta Virol 49, 159-67 (2005).-   141. Decman, V., Freeman, M. L., Kinchington, P. R. &    Hendricks, R. L. Immune control of HSV-1 latency. Viral Immunol 18,    466-73 (2005).-   142. Thompson, R. L. & Sawtell, N. M. The herpes simplex virus type    1 latency-associated transcript gene regulates the establishment of    latency. J Virol 71, 5432-40 (1997).-   143. Perng, G. C. et al. Virus-induced neuronal apoptosis blocked by    the herpes simplex virus latencyassociated transcript. Science 287,    1500-3 (2000).-   144. Thompson, R. L. & Sawtell, N. M. Herpes simplex virus type 1    latency-associated transcript gene promotes neuronal survival. J    Virol 75, 6660-75 (2001).-   145. Bhuyan, P. K. et al. Short interfering RNA-mediated inhibition    of herpes simplex virus type 1 gene expression and function during    infection of human keratinocytes. J Virol 78, 10276-81 (2004).-   146. Peng, W. et al. Mapping herpes simplex virus type 1    latency-associated transcript sequences that protect from apoptosis    mediated by a plasmid expressing caspase-8. J Neurovirol 10, 260-5    (2004).-   147. Jin, L. et al. Identification of herpes simplex virus type 1    latency-associated transcript sequences that both inhibit apoptosis    and enhance the spontaneous reactivation phenotype. J Virol 77,    6556-61 (2003).-   148. Wang, Q. Y. et al. Herpesviral latency-associated transcript    gene promotes assembly of heterochromatin on viral lytic-gene    promoters in latent infection. Proc Natl Acad Sci USA 102, 16055-9    (2005).-   149. Higaki, S., Deai, T., Fukuda, M. & Shimomura, Y. Microarray    analysis in the HSV-1 latently infected mouse trigeminal ganglion.    Cornea 23, S42-7 (2004).-   150. Jin, L. et al. A herpes simplex virus type 1 mutant expressing    a baculovirus inhibitor of apoptosis gene in place of    latency-associated transcript has a wild-type reactivation phenotype    in the mouse. J Virol 79, 12286-95 (2005).-   151. Sharma, S., Zhou, Y. & Singh, B. R. Cloning, expression, and    purification of C-terminal quarter of the heavy chain of botulinum    neurotoxin type A. Protein Expr Purif (2005).-   152. Zhou, Y. & Singh, B. R. Cloning, high-level expression,    single-step purification, and binding activity of His6-tagged    recombinant type B botulinum neurotoxin heavy chain transmembrane    and binding domain. Protein Expr Purif 34, 8-16 (2004).-   153. Lentz, T. L., Burrage, T. G., Smith, A. L., Crick, J. &    Tignor, G. H. Is the acetylcholine receptor a rabies virus receptor?    Science 215, 182-4 (1982).-   154. Lafon, M. Rabies virus receptors. J Neurovirol 11, 82-7 (2005).-   155. Rubinson, D. A. et al. A lentivirus-based system to    functionally silence genes in primary mammalian cells, stem cells    and transgenic mice by RNA interference. Nat Genet 33, 401-6 (2003).-   156. Mazarakis, N. D. et al. Rabies virus glycoprotein pseudotyping    of lentiviral vectors enables retrograde axonal transport and access    to the nervous system after peripheral delivery. Hum Mol Genet 10,    2109-21 (2001).-   157. Kumar, P., Lee, S. K., Shankar, P. & Manjunath, N. A Single    siRNA Suppresses Fatal Encephalitis Induced by Two Different    Flaviviruses. PLoS Med 3, e96 (2006).-   158. Judge, A. D. et al. Sequence-dependent stimulation of the    mammalian innate immune response by synthetic siRNA. Nat Biotechnol    23, 457-62 (2005).

1.-65. (canceled)
 66. A composition for targeted delivery of an effectoragent to a neuronal cell, astrocyte or microglia cell, comprising anamino acid sequence of SEQ ID NO: 13 or a variant of at least 85%sequence identity to SEQ ID NO: 1, and at least one targeting agent andat least one effector agent.
 67. The composition of claim 66, whereinthe effector agent is conjugated to the amino acid sequence of SEQ IDNO: 13 or a variant of at least 85% sequence identity to SEQ ID NO: 1.68. The composition of claim 66, wherein the amino acid sequence of SEQID NO: 13 or a variant of at least 85% sequence identity to SEQ ID NO: 1is present on the surface of a nanoparticle and wherein the effectoragent is present on the surface or in the nanoparticle.
 69. Thecomposition of claim 68, wherein the nanoparticle is a liposome orpolymeric nanoparticle.
 70. The composition of claim 69, wherein theliposome or polymeric nanoparticle is a cationic liposome.
 71. Thecomposition of claim 66, wherein the effector agent is selected from thegroup consisting of: a nucleic acid agent, a RNAi agent or a microRNAagent, a small molecule, a protein, peptide, aptamer.
 72. Thecomposition of claim 71, wherein the nucleic acid is selected from thegroup consisting of: RNA, siRNA, mRNA, tRNA, miRNA, shRNA orcombinations thereof.
 73. The composition of claim 71, wherein thenucleic acid is selected from the group consisting of: DNA, antisensenucleic acids, oligonucleic acids, peptide nucleic acid (PNA),pseudo-complementary PNA (pcPNA), locked nucleic acid (LNA), andantigomirs.
 74. The composition of claim 66, wherein the effector agentis an antibody, peptidomimetic monoclonal antibody or avimir.
 75. Thecomposition of claim 66, wherein the amino acid sequence of SEQ ID NO:13 or a variant of at least 85% sequence identity to SEQ ID NO: 1 isconjugated to a cell permeable peptide.
 76. The composition of claim 75,wherein the cell permeable peptide is a polymer consisting of arginineresidues or is a protamine peptide.
 77. A method for treatment of cancercomprising administering the subject with cancer the compositionaccording to claim 66.